Novel human G-protein coupled receptor, HGPRBMY8, expressed highly in brain

ABSTRACT

The present invention describes a newly discovered human G-protein coupled receptor and its encoding polynucleotide. Also described are expression vectors, host cells, agonists, antagonists, antisense molecules, and antibodies associated with the polynucleotide and/or polypeptide of the present invention. In addition, methods for treating, diagnosing, preventing, and screening for disorders associated with aberrant cell growth, neurological conditions, and diseases or disorders related to the brain are illustrated.

[0001] This application claims benefit to provisional application U.S.Serial No. 60/248,285, filed Nov. 14, 2000; to provisional applicationU.S. Serial No. 60/268,581, filed Feb. 14, 2001; to provisionalapplication U.S. Serial No. 60/308,285, filed Jul. 27, 2001; and toprovisional application U.S. Serial No. 60/317,166, filed Sep. 4, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to the fields of pharmacogenomics,diagnostics and patient therapy. More specifically, the presentinvention relates to methods of diagnosing and/or treating diseasesinvolving the Human G-Protein Coupled Receptor, HGPRBMY8.

BACKGROUND OF THE INVENTION

[0003] It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins arereferred to as proteins participating in pathways with G-proteins or PPGproteins. Some examples of these proteins include the GPC receptors,such as those for adrenergic agents and dopamine (Kobilka, B. K., etal., PNAS, 84:46-50 (1987); Kobilka, B. K., et al., Science, 238:650-656(1987); Bunzow, J. R., et al., Nature, 336:783-787 (1988)), G-proteinsthemselves, effector proteins, e.g., phospholipase C, adenylate cyclase,and phosphodiesterase, and actuator proteins, e.g., protein kinase A andprotein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)).

[0004] For example, in one form of signal transduction, the effect ofhormone binding is activation of an enzyme, adenylate cyclase, insidethe cell. Enzyme activation by hormones is dependent on the presence ofthe nucleotide GTP, and GTP also influences hormone binding. A G-proteinconnects the hormone receptors to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by hormone receptors. TheGTP-carrying form then binds to an activated adenylate cyclase.Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns theG-protein to its basal, inactive form. Thus, the G-protein serves a dualrole, as an intermediate that relays the signal from receptor toeffector, and as a clock that controls the duration of the signal.

[0005] The membrane protein gene superfamily of G-protein coupledreceptors has been characterized as having seven putative transmembranedomains. The domains are believed to represent transmembrane a-helicesconnected by extracellular or cytoplasmic loops. G-protein coupledreceptors include a wide range of biologically active receptors, such ashormone, viral, growth factor and neuroreceptors.

[0006] G-protein coupled receptors have been characterized as includingthese seven conserved hydrophobic stretches of about 20 to 30 aminoacids, connecting at least eight divergent hydrophilic loops. TheG-protein family of coupled receptors includes dopamine receptors, whichbind to neuroleptic drugs, used for treating psychotic and neurologicaldisorders. Other examples of members of this family include calcitonin,adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine,serotonin, histamine, thrombin, kinin, follicle stimulating hormone,opsins, endothelial differentiation gene-1 receptor, rhodopsins,odorant, cytomegalovirus receptors, etc.

[0007] Most G-protein coupled receptors have single conserved cysteineresidues in each of the first two extracellular loops which formdisulfide bonds that are believed to stabilize functional proteinstructure. The 7 transmembrane regions are designated as TM1, TM2, TM3,TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.

[0008] Phosphorylation and lipidation (palmitylation or farnesylation)of cysteine residues can influence signal transduction of some G-proteincoupled receptors. Most G-protein coupled receptors contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxyl terminus. For several G-protein coupled receptors, such as theβ-adrenoreceptor, phosphorylation by protein kinase A and/or specificreceptor kinases mediates receptor desensitization.

[0009] For some receptors, the ligand binding sites of G-protein coupledreceptors are believed to comprise a hydrophilic socket formed byseveral G-protein coupled receptors transmembrane domains, which socketis surrounded by hydrophobic residues of the G-protein coupledreceptors. The hydrophilic side of each G-protein coupled receptortransmembrane helix is postulated to face inward and form the polarligand-binding site. TM3 has been implicated in several G-proteincoupled receptors as having a ligand-binding site, such as including theTM3 aspartate residue. Additionally, TM5 serines, a TM6 asparagine andTM6 or TM7 phenylalanines or tyrosines are also implicated in ligandbinding.

[0010] G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson et al., Endoc. Rev., 10:317-331 (1989)).Different G-protein β-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

[0011] G-protein coupled receptors (GPCRs) are one of the largestreceptor superfamilies known. These receptors are biologically importantand malfunction of these receptors results in diseases such asAlzheimer's, Parkinson, diabetes, dwarfism, color blindness, retinalpigmentosa and asthma. GPCRs are also involved in depression,schizophrenia, sleeplessness, hypertension, anxiety, stress, renalfailure and in several other cardiovascular, metabolic, neural, oncologyand immune disorders (F. Horn and G. Vriend, J. Mol. Med., 76: 464-468(1998)). They have also been shown to play a role in HIV infection (Y.Feng et al., Science, 272: 872-877 (1996)). The structure of GPCRsconsists of seven transmembrane helices that are connected by loops. TheN-terminus is always extracellular and C-terminus is intracellular.GPCRs are involved in signal transduction. The signal is received at theextracellular N-terminus side. The signal can be an endogenous ligand, achemical moiety or light. This signal is then transduced through themembrane to the cytosolic side where a heterotrimeric protein G-proteinis activated which in turn elicits a response (F. Horn et al., Recept.and Chann., 5: 305-314 (1998)). Ligands, agonists and antagonists, forthese GPCRs are used for therapeutic purposes.

[0012] The present invention provides a newly discovered G-proteincoupled receptor protein, which may be involved in cellular growthproperties in brain-related tissues based on its abundance found in thebrain for this receptor. The present invention also relates to newlyidentified polynucleotides, polypeptides encoded by suchpolynucleotides, the use of such polynucleotides and polypeptides, aswell as the production of such polynucleotides and polypeptides. Moreparticularly, the polypeptides of the present invention are human7-transmembrane receptors. The invention also relates to inhibiting theaction of such polypeptides.

SUMMARY OF THE INVENTION

[0013] The present invention provides a novel human member of theG-protein coupled receptor (GPCR) family (HGPRBMY8). Based on sequencehomology, the protein HGPRBMY8 is a candidate GPCR. Based on its proteinsequence information, the HGPRBMY8 contains seven transmembrane domains,which is a characteristic structural feature of GPCRs. The GPCR of thisinvention is closely related to the somatostatin and GPR24 receptorfamilies based on sequence similarity using the BLAST program. Thisorphan GPCR is expressed highly in brain.

[0014] It is an object of the present invention to provide an isolatedHGPRBMY8 polynucleotide as depicted in SEQ ID NO:1.

[0015] It is also an object of the present invention to provide theHGPRBMY8 polypeptide, encoded by the polynucleotide of SEQ ID NO:1(CDS=1 to 1524) and having the amino acid sequence of SEQ ID NO:2, or afunctional or biologically active portion thereof.

[0016] It is a further object of the present invention to providecompositions comprising the HGPRBMY8 polynucleotide sequence, or afragment thereof, or the encoded HGPRBMY8 polypeptide (MW=56.7 Kd), or afragment or portion thereof. Also provided by the present invention arepharmaceutical compositions comprising at least one HGPRBMY8polypeptide, or a functional portion thereof, wherein the compositionsfurther comprise a pharmaceutically acceptable carrier, excipient, ordiluent.

[0017] It is an object of the present invention to provide a novel,isolated, and substantially purified polynucleotide that encodes theHGPRBMY8 GPCR homologue, or fragment thereof. In a particular aspect,the polynucleotide comprises the nucleotide sequence of SEQ ID NO:1. Thepresent invention also provides a polynucleotide sequence comprising thecomplement of SEQ ID NO:1, or variants thereof. In addition, the presentinvention features polynucleotide sequences, which hybridize underconditions of moderate stringency or high stringency to thepolynucleotide sequence of SEQ ID NO:1.

[0018] It is an object of the present invention to further provide anucleic acid sequence encoding the HGPRBMY8 polypeptide and an antisenseof the nucleic acid sequence, as well as oligonucleotides, fragments, orportions of the nucleic acid molecule or antisense molecule. Alsoprovided are expression vectors and host cells comprisingpolynucleotides that encode the HGPRBMY8 polypeptide.

[0019] It is an object of the invention to provide methods for producinga polypeptide comprising the amino acid sequence depicted in SEQ IDNO:2, or a fragment thereof, comprising the steps of a) cultivating ahost cell containing an expression vector containing at least afunctional fragment of the polynucleotide sequence encoding the HGPRBMY8protein according to this invention under conditions suitable for theexpression of the encoded polypeptide; and b) recovering the polypeptidefrom the host cell.

[0020] It is also an object of the invention to provide antibodies, andbinding fragments thereof, which bind specifically to the HGPRBMY8polypeptide, or an epitope thereof, for use as therapeutic anddiagnostic agents.

[0021] It is a further object of the invention to provide methods forscreening for agents which bind to, or modulate HGPRBMY8 polypeptide,e.g., agonists and antagonists, as well as the binding molecules and/ormodulators, e.g., agonists and antagonists, particularly those that areobtained from the screening methods described.

[0022] It is an object of the present invention to also provide asubstantially purified antagonist or inhibitor of the polypeptide of SEQID NO:2. In this regard, and by way of example, a purified antibody thatbinds to a polypeptide comprising the amino acid sequence of SEQ ID NO:2is provided.

[0023] It is an object of the invention to further provide substantiallypurified agonists or activators of the polypeptide of SEQ ID NO:2 arefurther provided.

[0024] It is another object of the present invention to provide HGPRBMY8nucleic acid sequences, polypeptide, peptides and antibodies for use inthe diagnosis and/or screening of disorders or diseases associated withexpression of the polynucleotide and its encoded polypeptide asdescribed herein.

[0025] It is a also an object of the present invention to provide kitsfor screening and diagnosis of disorders associated with aberrant oruncontrolled cellular development and with the expression of thepolynucleotide and its encoded polypeptide as described herein.

[0026] It is an object of the present invention to further providemethods for the treatment or prevention of cancers, immune disorders, orneurological disorders involving administering to an individual in needof treatment or prevention an effective amount of a purified antagonistof the HGPRBMY8 polypeptide. Due to its elevated expression in brain,the novel GPCR protein of the present invention is particularly usefulin treating or preventing neurological disorders, conditions, ordiseases.

[0027] It is an object of the present invention to also provide a methodfor detecting a polynucleotide that encodes a G-protein coupledreceptor, preferably the HGPRBMY8 polypeptide, or homologue, or fragmentthereof, in a biological sample comprising the steps of: a) hybridizingthe polynucleotide, or complement of the polynucleotide sequenceencoding SEQ ID NO:2 to a nucleic acid material of a biological sample,thereby forming a hybridization complex; and b) detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding the HGPRBMY8 polypeptide,or fragment therof, in the biological sample. The nucleic acid materialmay be further amplified by the polymerase chain reaction prior tohybridization.

[0028] It is an object of the instant invention to provide methods andcompositions to detect and diagnose alterations in the HGPRBMY8 sequencein tissues and cells as they relate to ligand response.

[0029] It is an object of the present invention to further providecompositions for diagnosing brain-related disorders and for diagnosingor monitoring response to HGPRBMY8 therapy in humans. In accordance withthe invention, the compositions detect an alteration of the normal orwild type HGPRBMY8 sequence or its expression product in a patientsample of cells or tissue.

[0030] It is an object of the present invention to provide diagnosticprobes for diseases and a patient's response to therapy. The probesequence comprises the HGPRBMY8 locus polymorphism. The probes can beconstructed of nucleic acids or amino acids.

[0031] It is an object of the present invention to further provideantibodies, and immunoreactive portions thereof, that recognize and bindto the HGPRBMY8 protein. Such antibodies can be either polyclonal ormonoclonal. Antibodies that bind to the HGPRBMY8 protein can be utilizedin a variety of diagnostic and prognostic formats and therapeuticmethods.

[0032] It is also an object of the present invention to providediagnostic kits for the determination of the nucleotide sequence ofhuman HGPRBMY8 alleles. The kits are based on amplification-basedassays, nucleic acid probe assays, protein nucleic acid probe assays,antibody assays or any combination thereof.

[0033] It is an object of the instant invention to further providemethods for detecting genetic predisposition, susceptibility andresponse to therapy related to the brain. In accordance with theinvention, the method comprises isolating a human sample, for example,blood or tissue from adults, children, embryos or fetuses, and detectingat least one alteration in the wild type HGPRBMY8 sequence, or itsexpression product, from the sample, wherein the alterations areindicative of genetic predisposition, susceptibility or altered responseto therapy related to the brain.

[0034] It is an additional object of the present invention to providemethods for making determinations as to which drug to administer,dosages, duration of treatment and the like.

[0035] Further objects, features, and advantages of the presentinvention will be better understood upon a reading of the detaileddescription of the invention when considered in connection with theaccompanying figures/drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0036]FIG. 1 shows the full-length nucleotide sequence of cDNA cloneHGPRBMY8, a human G-protein coupled receptor (SEQ ID NO:1).

[0037]FIG. 2 shows the amino acid sequence (SEQ ID NO:2) from thetranslation of the full-length HGPRBMY8 cDNA sequence.

[0038]FIG. 3 shows the 5′ untranslated sequence of the orphan HGPRBMY8(SEQ ID NO:3).

[0039]FIG. 4 shows the 3′ untranslated sequence of the orphan HGPRBMY8(SEQ ID NO:4).

[0040]FIG. 5 shows the predicted transmembrane region of the HGPRBMY8protein where the predicted transmembrane regions, represented bybold-faced and underlined type, correspond to the peaks with scoresabove 1500.

[0041] FIGS. 6A-6J show the multiple sequence alignment of thetranslated sequence of the orphan G-protein coupled receptor, HGPRBMY8,where the GCG (Genetics Computer Group) pileup program was used togenerate the alignment with several known adrenergic and serotoninreceptor sequences. The blackened areas represent identical amino acidsin more than half of the listed sequences and the grey highlighted areasrepresent similar amino acids. As shown in FIGS. 6A-6J, the sequencesare aligned according to their amino acids, where: HGPRBMY8 (SEQ IDNO:2) is encoded by full length HGPRBMY8 cDNA; ACM4_CHICK (SEQ ID NO:7)represents the Gallus gallus (chicken) form of muscarinic acetylcholinereceptor M4; YDBM_CAEEL (SEQ ID NO:8) is the Caenorhabditis elegans formof an orphan GPCR; 5H1A_HUMAN (SEQ ID NO:9) is the human form of the5HT-1A receptor; 5H1A_MOUSE (SEQ ID NO:10) is the Mus musculus (housemouse) form of the 5HT-1A receptor; 5H1A_FUGRU (SEQ ID NO:11) representsthe Fugu rubripes form of the 5HT-1A receptor; 5HT_LYMST (SEQ ID NO:12)is the Lymnaea stagnalis (great pond snail) form of the 5HT-1A receptor;A1AD_HUMAN (SEQ ID NO:13) is the human form of the alpha-1D adrenergicreceptor; A1AD_MOUSE (SEQ ID NO:14) represents the mouse form of thealpha-1D adrenergic receptor (alpha 1D-adrenoceptor); Q13675 (SEQ IDNO:15) is the human form of the alpha 1C adrenergic receptor isoform 2;Q13729 (SEQ ID NO:16) represents the human form of the alpha 1Cadrenergic receptor isoform 3; O60451 is the human form of the alpha 1Aadrenergic receptor isoform 4 (SEQ ID NO:17); A1AA_RAT (SEQ ID NO:18) isthe Rattus norvegicus (Norway rat) form of the alpha-1A adrenergicreceptor; O54913 (SEQ ID NO:19) is the Mus musculus (house mouse) formof the alpha 1A-adrenergic receptor; A1AA_BOVIN (SEQ ID NO:20)represents the Bos taurus (bovine) form of the alpha-1A adrenergicreceptor; A1AA_CANFA (SEQ ID NO:21) is the Canis familiaris (dog) formof the alpha-1A adrenergic receptor; A1AA_RABIT (SEQ ID NO:22)represents the Oryctolagus cuniculus (rabbit) form of the alpha-1Aadrenergic receptor; A1AA_HUMAN (SEQ ID NO:23) is the human form of thealpha-1A adrenergic receptor; A1AA_ORYLA (SEQ ID NO:24) is the Oryziaslatipes (japanese medaka) form of the alpha-1A adrenergic receptor(MAR1); and O96716 (SEQ ID NO:25) represents the Branchiostomalanceolatum (amphioxus) form of the dopamine D1/beta receptor; andO75963 (SEQ ID NO:40) is the human form of the G-protein coupledreceptor RE2.

[0042]FIG. 7 shows the expression profiling of the novel human orphanGPCR, HGPRBMY8, as described in Example 3.

[0043]FIG. 8 shows the brain-specific expression profiling of the novelhuman orphan GPCR, HGPRBMY8, as described in Example 4.

[0044]FIG. 9 shows the multiple sequence alignment of HGPRBMY8 and otherpotential SNP variants (amino acid alignment). The blackened areasrepresent identical amino acids and the grey highlighted areas representsimilar amino acids. As shown in FIG. 9, the sequences are alignedaccording to their amino acids, where: AL390879 (SEQ ID NO:41), AX148250(SEQ ID NO:42), and AX080495 (SEQ ID NO:43) are compared to HGPRBMY8(SEQ ID NO:2).

[0045] FIGS. 10A-D shows the multiple sequence alignment of HGPRBMY8 andother potential SNP variants (nucleic acid alignment). The blackenedareas represent identical amino acids and the grey highlighted areasrepresent similar amino acids. As shown in FIG. 10, the sequences arealigned according to their nucleic acids, where: AX080495 (SEQ IDNO:44); AL390879 (SEQ ID NO:45), AX148250 (SEQ ID NO:46), and arecompared to HGPRBMY8 (SEQ ID NO:47).

[0046]FIG. 11 shows the FACS profile of an untransfected CHO-NFAT/CREcell line.

[0047]FIG. 12 shows that overexpression of HGPRBMY8 constitutivelycouples through the NFAT/CRE Response Element.

[0048]FIG. 13 shows the FACS profile for the untransfected cAMP ResponseElement.

[0049]FIG. 14 shows the overexpression of HGPRBMY8 results in couplingthrough the cAMP Response Element.

[0050] FIGS. 15A-D shows the localization of expressed HGPRBMY8 to thecell surface.

[0051] FIGS. 16A-D shows representative transfected CHO-NFAT/CRE celllines with intermediate and high beta lactamase expression levels usefulin screens to identify HGPRBMY8 agonists and/or antagonists.

[0052]FIG. 17 shows the expression profiling of the novel human orphanGPCR, HGPRBMY8, as described in Example 8 and Table 1.

[0053] FIGS. 18A-B show the polynucleotide sequence (SEQ ID NO:48) anddeduced amino acid sequence (SEQ ID NO:49) of the human G-proteincoupled receptor, HGPRBMY8, comprising, or alternatively consisting of,one or more of the preducted polynucleotide polymorphic loci, inaddition to, the encoded polypeptide polymorphic loci of the presentinvention for this particular protein.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0054] The present invention provides a novel isolated polynucleotideand encoded polypeptide, the expression of which is high in brain. Thisnovel polypeptide is termed herein HGPRBMY8, an acronym for “HumanG-Protein coupled Receptor BMY8”. HGPRBMY8 is also referred to as GPCR58and GPCR84.

Definitions

[0055] The HGPRBMY8 polypeptide (or protein) refers to the amino acidsequence of substantially purified HGPRBMY8, which may be obtained fromany species, preferably mammalian, and more preferably, human, and froma variety of sources, including natural, synthetic, semi-synthetic, orrecombinant. Functional fragments of the HGPRBMY8 polypeptide are alsoembraced by the present invention.

[0056] An “agonist” refers to a molecule which, when bound to theHGPRBMY8 polypeptide, or a functional fragment thereof, increases orprolongs the duration of the effect of the HGPRBMY8 polypeptide.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules that bind to and modulate the effect of HGPRBMY8polypeptide. An antagonist refers to a molecule which, when bound to theHGPRBMY8 polypeptide, or a functional fragment thereof, decreases theamount or duration of the biological or immunological activity ofHGPRBMY8 polypeptide. “Antagonists” may include proteins, nucleic acids,carbohydrates, antibodies, or any other molecules that decrease orreduce the effect of HGPRBMY8 polypeptide.

[0057] “Nucleic acid sequence”, as used herein, refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or anti-sensestrand. By way of non-limiting example, fragments include nucleic acidsequences that are greater than 20-60 nucleotides in length, andpreferably include fragments that are at least 70-100 nucleotides, orwhich are at least 1000 nucleotides or greater in length.

[0058] Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.Amino acid sequence fragments are typically from about 5 to about 30,preferably from about 5 to about 15 amino acids in length and retain thebiological activity or function of the HGPRBMY8 polypeptide.

[0059] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. In addition,the terms HGPRBMY8 polypeptide and HGPRBMY8 protein are usedinterchangeably herein to refer to the encoded product of the HGPRBMY8nucleic acid sequence of the present invention.

[0060] A “variant” of the HGPRBMY8 polypeptide refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “non-conservative”changes, e.g., replacement of a glycine with a tryptophan. Minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing functional biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0061] An “allele” or “allelic sequence” is an alternative form of theHGPRBMY8 nucleic acid sequence. Alleles may result from at least onemutation in the nucleic acid sequence and may yield altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene, whether natural or recombinant, may have none, one, or manyallelic forms. Common mutational changes, which give rise to alleles,are generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0062] “Altered” nucleic acid sequences encoding HGPRBMY8 polypeptideinclude nucleic acid sequences containing deletions, insertions and/orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent HGPRBMY8 polypeptide.Altered nucleic acid sequences may further include polymorphisms of thepolynucleotide encoding the HGPRBMY8 polypeptide; such polymorphisms mayor may not be readily detectable using a particular oligonucleotideprobe. The encoded protein may also contain deletions, insertions, orsubstitutions of amino acid residues, which produce a silent change andresult in a functionally equivalent HGPRBMY8 protein. Deliberate aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological activityof HGPRBMY8 protein is retained. For example, negatively charged aminoacids may include aspartic acid and glutamic acid; positively chargedamino acids may include lysine and arginine; and amino acids withuncharged polar head groups having similar hydrophilicity values mayinclude leucine, isoleucine, and valine; glycine and alanine; asparagineand glutamine; serine and threonine; and phenylalanine and tyrosine.

[0063] “Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide (“oligo”) linked viaan amide bond, similar to the peptide backbone of amino acid residues.PNAs typically comprise oligos of at least 5 nucleotides linked viaamide bonds. PNAs may or may not terminate in positively charged aminoacid residues to enhance binding affinities to DNA. Such amino acidsinclude, for example, lysine and arginine, among others. These smallmolecules stop transcript elongation by binding to their complementarystrand of nucleic acid (P. E. Nielsen et al., 1993, Anticancer DrugDes., 8:53-63). PNA may be pegylated to extend their lifespan in thecell where they preferentially bind to complementary single stranded DNAand RNA.

[0064] “Oligonucleotides” or “oligomers” refer to a nucleic acidsequence, preferably comprising contiguous nucleotides, of at leastabout 6 nucleotides to about 60 nucleotides, preferably at least about 8to 10 nucleotides in length, more preferably at least about 12nucleotides in length e.g., about 15 to 35 nucleotides, or about 15 to25 nucleotides, or about 20 to 35 nucleotides, which can be typicallyused in PCR amplification assays, hybridization assays, or inmicroarrays. It will be understood that the term oligonucleotide issubstantially equivalent to the terms primer, probe, or amplimer, ascommonly defined in the art. It will also be appreciated by thoseskilled in the pertinent art that a longer oligonucleotide probe, ormixtures of probes, e.g., degenerate probes, can be used to detectlonger, or more complex, nucleic acid sequences, for example, genomicDNA. In such cases, the probe may comprise at least 20-200 nucleotides,preferably, at least 30-100 nucleotides, more preferably, 50-100nucleotides.

[0065] “Amplification” refers to the production of additional copies ofa nucleic acid sequence and is generally carried out using polymerasechain reaction (PCR) technologies, which are well known and practiced inthe art (see, D. W. Dieffenbach and G. S. Dveksler, 1995, PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0066] “Microarray” is an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon, orother type of membrane; filter; chip; glass slide; or any other type ofsuitable solid support.

[0067] The term “antisense” refers to nucleotide sequences, andcompositions containing nucleic acid sequences, which are complementaryto a specific DNA or RNA sequence. The term “antisense strand” is usedin reference to a nucleic acid strand that is complementary to the“sense” strand. Antisense (i.e., complementary) nucleic acid moleculesinclude PNA and may be produced by any method, including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes, which block either transcription or translation. Thedesignation “negative” is sometimes used in reference to the antisensestrand, and “positive” is sometimes used in reference to the sensestrand.

[0068] The term “consensus” refers to the sequence that reflects themost common choice of base or amino acid at each position among a seriesof related DNA, RNA or protein sequences. Areas of particularly goodagreement often represent conserved functional domains.

[0069] A “deletion” refers to a change in either nucleotide or aminoacid sequence and results in the absence of one or more nucleotides oramino acid residues. By contrast, an insertion (also termed “addition”)refers to a change in a nucleotide or amino acid sequence that resultsin the addition of one or more nucleotides or amino acid residues, ascompared with the naturally occurring molecule. A substitution refers tothe replacement of one or more nucleotides or amino acids by differentnucleotides or amino acids.

[0070] A “derivative” nucleic acid molecule refers to the chemicalmodification of a nucleic acid encoding, or complementary to, theencoded HGPRBMY8 polypeptide. Such modifications include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A nucleicacid derivative encodes a polypeptide, which retains the essentialbiological and/or functional characteristics of the natural molecule. Aderivative polypeptide is one, which is modified by glycosylation,pegylation, or any similar process that retains the biological and/orfunctional or immunological activity of the polypeptide from which it isderived.

[0071] The term “biologically active”, i.e., functional, refers to aprotein or polypeptide or fragment thereof having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” refers to the capability of thenatural, recombinant, or synthetic HGPRBMY8, or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells, for example, to generate antibodies, and to bind with specificantibodies.

[0072] The term “hybridization” refers to any process by which a strandof nucleic acid binds with a complementary strand through base pairing.

[0073] The term “hybridization complex” refers to a complex formedbetween two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases. The hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an anti-parallel configuration. Ahybridization complex may be formed in solution (e.g., C_(o)t or R_(o)tanalysis), or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins, or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenaffixed).

[0074] The terms “stringency” or “stringent conditions” refer to theconditions for hybridization as defined by nucleic acid composition,salt and temperature. These conditions are well known in the art and maybe altered to identify and/or detect identical or related polynucleotidesequences in a sample. A variety of equivalent conditions comprisingeither low, moderate, or high stringency depend on factors such as thelength and nature of the sequence (DNA, RNA, base composition), reactionmilieu (in solution or immobilized on a solid substrate), nature of thetarget nucleic acid (DNA, RNA, base composition), concentration of saltsand the presence or absence of other reaction components (e.g.,formamide, dextran sulfate and/or polyethylene glycol) and reactiontemperature (within a range of from about 5° C. below the meltingtemperature of the probe to about 20° C. to 25° C. below the meltingtemperature). One or more factors may be varied to generate conditions,either low or high stringency that is different from but equivalent tothe aforementioned conditions.

[0075] As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences. As will be furtherappreciated by the skilled practitioner, the melting temperature, T_(m),can be approximated by the formulas as known in the art, depending on anumber of parameters, such as the length of the hybrid or probe innumber of nucleotides, or hybridization buffer ingredients andconditions (see, for example, T. Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1982 and J. Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Current Protocols in Molecular Biology, Eds. F. M. Ausubel et al., Vol.1, “Preparation and Analysis of DNA”, John Wiley and Sons, Inc.,1994-1995, Suppls. 26, 29, 35 and 42; pp. 2.10.7-2.10.16; G. M. Wahl andS. L. Berger (1987; Methods Enzymol. 152:399-407); and A. R. Kimmel,1987; Methods of Enzymol. 152:507-511). As a general guide, T_(m)decreases approximately 1° C.-1.5° C. with every 1% decrease in sequencehomology. Also, in general, the stability of a hybrid is a function ofsodium ion concentration and temperature. Typically, the hybridizationreaction is initially performed under conditions of low stringency,followed by washes of varying, but higher stringency. Reference tohybridization stringency, e.g., high, moderate, or low stringency,typically relates to such washing conditions.

[0076] Thus, by way of non-limiting example, “high stringency” refers toconditions that permit hybridization of those nucleic acid sequencesthat form stable hybrids in 0.018M NaCl at about 65° C. (i.e., if ahybrid is not stable in 0.018M NaCl at about 65° C., it will not bestable under high stringency conditions). High stringency conditions canbe provided, for instance, by hybridization in 50% formamide, 5×Denhardt's solution, 5× SSPE (saline sodium phosphate EDTA) (1× SSPEbuffer comprises 0.15 M NaCl, 10 mM Na₂HPO₄, 1 mM EDTA), (or 1× SSCbuffer containing 150 mM NaCl, 15 mM Na₃ citrate•2 H₂O, pH 7.0), 0.2%SDS at about 42° C., followed by washing in 1× SSPE (or saline sodiumcitrate, SSC) and 0.1% SDS at a temperature of at least about 42° C.,preferably about 55° C., more preferably about 65° C.

[0077] “Moderate stringency” refers, by non-limiting example, toconditions that permit hybridization in 50% formamide, 5× Denhardt'ssolution, 5× SSPE (or SSC), 0.2% SDS at 42° C. (to about 50° C.),followed by washing in 0.2× SSPE (or SSC) and 0.2% SDS at a temperatureof at least about 42° C., preferably about 55° C., more preferably about65° C.

[0078] “Low stringency” refers, by non-limiting example, to conditionsthat permit hybridization in 10% formamide, 5× Denhardt's solution, 6×SSPE (or SSC), 0.2% SDS at 42° C., followed by washing in 1× SSPE (orSSC) and 0.2% SDS at a temperature of about 45° C., preferably about 50°C.

[0079] For additional stringency conditions, see T. Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). It is to be understood that the low,moderate and high stringency hybridization/washing conditions may bevaried using a variety of ingredients, buffers and temperatures wellknown to and practiced by the skilled artisan.

[0080] The terms “complementary” or “complementarity” refer to thenatural binding of polynucleotides under permissive salt and temperatureconditions by base pairing. For example, the sequence “A-G-T” binds tothe complementary sequence “T-C-A”. Complementarity between twosingle-stranded molecules may be “partial”, in which only some of thenucleic acids bind, or it may be complete when total complementarityexists between single stranded molecules. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, which depend uponbinding between nucleic acids strands, as well as in the design and useof PNA molecules.

[0081] The term “homology” refers to a degree of complementarity. Theremay be partial homology or complete homology, wherein complete homologyis equivalent to identity. A partially complementary sequence that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid is referred to using the functional term“substantially homologous”. The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (e.g., Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. Nonetheless, conditions of low stringency do not permitnon-specific binding; low stringency conditions require that the bindingof two sequences to one another be a specific (i.e., selective)interaction. The absence of non-specific binding may be tested by theuse of a second target sequence which lacks even a partial degree ofcomplementarity (e.g., less than about 30% identity). In the absence ofnon-specific binding, the probe will not hybridize to the secondnon-complementary target sequence.

[0082] Those having skill in the art will know how to determine percentidentity between or among sequences using, for example, algorithms suchas those based on the CLUSTALW computer program (J. D. Thompson et al.,1994, Nucleic Acids Research, 2(22):4673-4680), or FASTDB, (Brutlag etal., 1990, Comp. App. Biosci., 6:237-245), as known in the art. Althoughthe FASTDB algorithm typically does not consider internal non-matchingdeletions or additions in sequences, i.e., gaps, in its calculation,this can be corrected manually to avoid an overestimation of the %identity. CLUSTALW, however, does take sequence gaps into account in itsidentity calculations.

[0083] A “composition” comprising a given polynucleotide sequence refersbroadly to any composition containing the given polynucleotide sequence.The composition may comprise a dry formulation or an aqueous solution.Compositions comprising polynucleotide sequence (SEQ ID NO:1) encodingHGPRBMY8 polypeptide (SEQ ID NO:2), or fragments thereof, may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be in association with a stabilizing agentsuch as a carbohydrate. In hybridizations, the probe may be employed inan aqueous solution containing salts (e.g., NaCl), detergents orsurfactants (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, and the like).

[0084] The term “substantially purified” refers to nucleic acidsequences or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% to 85% free, and most preferably 90% or greater free fromother components with which they are naturally associated.

[0085] The term “sample”, or “biological sample”, is meant to beinterpreted in its broadest sense. A biological sample suspected ofcontaining nucleic acid encoding HGPRBMY8 protein, or fragments thereof,or HGPRBMY8 protein itself, may comprise a body fluid, an extract fromcells or tissue, chromosomes isolated from a cell (e.g., a spread ofmetaphase chromosomes), organelle, or membrane isolated from a cell, acell, nucleic acid such as genomic DNA (in solution or bound to a solidsupport such as for Southern analysis), RNA (in solution or bound to asolid support such as for Northern analysis), cDNA (in solution or boundto a solid support), a tissue, a tissue print and the like.

[0086] “Transformation” refers to a process by which exogenous DNAenters and changes a recipient cell. It may occur under natural orartificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the type of host cell being transformed andmay include, but is not limited to, viral infection, electroporation,heat shock, lipofection, and partial bombardment. Such “transformed”cells include stably transformed cells in which the inserted DNA iscapable of replication either as an autonomously replicating plasmid oras part of the host chromosome. Transformed cells also include thosecells, which transiently express the inserted DNA or RNA for limitedperiods of time.

[0087] The term “mimetic” refers to a molecule, the structure of whichis developed from knowledge of the structure of HGPRBMY8 protein, orportions thereof, and as such, is able to effect some or all of theactions of HGPRBMY8 protein.

[0088] The term “portion” with regard to a protein (as in “a portion ofa given protein”) refers to fragments or segments of that protein. Thefragments may range in size from four or five amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:2” encompasses the full-length human HGPRBMY8 polypeptide, andfragments thereof.

[0089] The term “antibody” refers to intact molecules as well asfragments thereof, such as Fab, F(ab′)₂, Fv, or Fc, which are capable ofbinding an epitopic or antigenic determinant. Antibodies that bind toHGPRBMY8 polypeptides can be prepared using intact polypeptides orfragments containing small peptides of interest or preparedrecombinantly for use as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal can be derived from thetransition of RNA or synthesized chemically, and can be conjugated to acarrier protein, if desired. Commonly used carriers that are chemicallycoupled to peptides include, but are not limited to, bovine serumalbumin (BSA), keyhole limpet hemocyanin (KLH), and thyroglobulin. Thecoupled peptide is then used to immunize the animal (e.g, a mouse, arat, or a rabbit).

[0090] The term “humanized” antibody refers to antibody molecules inwhich amino acids have been replaced in the non-antigen binding regionsin order to more closely resemble a human antibody, while stillretaining the original binding capability, e.g., as described in U.S.Pat. No. 5,585,089 to C. L. Queen et al.

[0091] The term “antigenic determinant” refers to that portion of amolecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to an antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0092] The terms “specific binding” or “specifically binding” refer tothe interaction between a protein or peptide and a binding molecule,such as an agonist, an antagonist, or an antibody. The interaction isdependent upon the presence of a particular structure (i.e., anantigenic determinant or epitope) of the protein that is recognized bythe binding molecule. For example, if an antibody is specific forepitope “A”, the presence of a protein containing epitope A (or free,unlabeled A) in a reaction containing labeled “A” and the antibody willreduce the amount of labeled A bound to the antibody.

[0093] The term “correlates with expression of a polynucleotide”indicates that the detection of the presence of ribonucleic acid that issimilar to SEQ ID NO:1 by Northern analysis is indicative of thepresence of mRNA encoding HGPRBMY8 polypeptide (SEQ ID NO:2) in a sampleand thereby correlates with expression of the transcript from thepolynucleotide encoding the protein.

[0094] An alteration in the polynucleotide of SEQ ID NO:1 comprises anyalteration in the sequence of the polynucleotides encoding HGPRBMY8polypeptide, including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes HGPRBMY8 polypeptide (e.g., by alterations in the patternof restriction fragment length polymorphisms capable of hybridizing toSEQ ID NO:1), the inability of a selected fragment of SEQ ID NO:1 tohybridize to a sample of genomic DNA (e.g., using allele-specificoligonucleotide probes), and improper or unexpected hybridization, suchas hybridization to a locus other than the normal chromosomal locus forthe polynucleotide sequence encoding HGPRBMY8 polypeptide (e.g., usingfluorescent in situ hybridization (FISH) to metaphase chromosomespreads).

DESCRIPTION OF THE PRESENT INVENTION

[0095] The present invention provides a novel human member of theG-protein coupled receptor (GPCR) family (HGPRBMY8). Based on sequencehomology, the protein HGPRBMY8 is a novel human GPCR. This proteinsequence has been predicted to contain seven transmembrane domains whichis a characteristic structural feature of GPCRs. HGPRBMY8 belongs to the“class A” of GPCR superfamily and is closely related to adrenergic andserotonin receptors based on sequence similarity. Class A is the largestsub-family of the GPCR superfamily. This particular orphan GPCR isexpressed highly in brain.

[0096] HGPRBMY8 polypeptides and polynucleotides are useful fordiagnosing diseases related to over- or under-expression of HGPRBMY8proteins by identifying mutations in the HGPRBMY8 gene using HGPRBMY8probes, or by determining HGPRBMY8 protein or mRNA expression levels.HGPRBMY8 polypeptides are also useful for screening compounds, whichaffect activity or function of the protein. The invention encompassesthe polynucleotide encoding the HGPRBMY8 polypeptide and the use of theHGPRBMY8 polynucleotide or polypeptide, or composition thereof, in thescreening, diagnosis, treatment, or prevention of disorders associatedwith aberrant or uncontrolled cellular growth and/or function, such asneoplastic diseases (e.g., cancers and tumors), with particular regardto diseases or disorders related to the brain, e.g. neurologicaldisorders.

[0097] Nucleic acids encoding human HGPRBMY8 according to the presentinvention were first identified from the human genomic data availablefrom GenBank (Accession No: AC016468).

[0098] In one of its embodiments, the present invention encompasses apolypeptide comprising the amino acid sequence of SEQ ID NO:2 as shownin FIG. 1. The HGPRBMY8 polypeptide is 508 amino acids in length andshares amino acid sequence homology with the GPCR RE2. The HGPRBMY8polypeptide (SEQ ID NO:2) shares 24.3 % identity and 33.6 % similaritywith over 400 amino acids of the GPCR RE2 sequence, wherein “similar”amino acids are those which have the same/similar physical propertiesand in many cases, the function is conserved with similar residues. Forexample, amino acids Lysine and Arginine are similar; while residuessuch as Proline and Cysteine, which do not share any physicalproperties, are considered dissimilar. The HGPRBMY8 polypeptide shares28.01% identity and 38.33% similarity with the Fugu rubripes5-Hydroxytryptamine 1a-Alpha Receptor (5H1A_FUGRU; Acc. No.:O42385);25.3% identity and 37.23% similarity with the human 5-Hydroxytryptamine1a-Alpha Receptor (5H1A_HUMAN; Acc. No.:P08908); 27.56% idenity and37.56% similarity with the Mus musculus 5-Hydroxytryptamine 1a-AlphaReceptor (5H1A_MOUSE; Acc. No.:Q64264, Q60956); 25.46% identity and37.05% similarity with the Lymnaea stagnalis 5-hydroxytryptaminereceptor (5HT_LYMST; Acc. No.:Q25414); 23.67% identity and 33.19%similarity with the Bos taurus Alpha-1A adrenergic receptor (A1AA_BOVIN;Acc. No.: P18130); 26.21% identity and 36.9% similarity with the Canisfamiliaris Alpha-1A adrenergic receptor (A1AA_CANFA; Acc. No.:O77621);29.47% identity and 41.05% similarity with the human Alpha-1A adrenergicreceptor (A1AA_HUMAN; Acc. No.: P35348); 31.65% identity and 42.29%similarity with the Oryzias latipes Alpha-1A adrenergic receptor(A1AA_ORYLA; Acc. No.:Q91175); 30% identity and 41.32% similarity withthe Oryctolagus cuniculus Alpha-1A adrenergic receptor (A1AA_RABIT; Acc.No.:O02824); 24.82% identity and 34.43% similarity with the Rattusnorvegicus Alpha-1A adrenergic receptor (A1AA_RAT; Acc. No.:P43140);29.79% identity and 41.19% similarity with the human Alpha-1D adrenergicreceptor (A1AD_HUMAN; Acc. No.: P25100); 29.2% identity and 40.57%similarity with the Mus musculus Alpha-1D adrenergic receptor(A1AD_MOUSE; Acc. No.:P97714, Q61619); 23.33% identity and 31.97%similarity with the Gallus gallus muscarinic acetylcholine receptor M4(ACM4_CHICK; Acc. No.:P17200); 30.53% identity and 41.58% similaritywith the Mus musculus Alpha-1A adrenergic receptor (O54913; Acc.No.:O54913); 29.47% identity and 41.05% similarity with the humanAlpha-1A adrenergic receptor isoform 4 (O60451; Acc. No.:O60451); 23.59%identity and 32.82% similarity with the human G-protein coupled receptorRE2 (O75963; Acc. No.:O75963); 23.99% identity and 31.81% similaritywith the Branchiostoma lanceolatum dopamine D1/Beta receptor (O96716;Acc. No.:O96716); 29.21% identity and 40.79% similarity with the humanAlpha 1C adrenergic receptor isoform 2 (Q13675; Acc. No.:Q13675); 24.87%identity and 34.52% similarity with the human Alpha 1C adrenergicreceptor isoform 3 (Q13729; Acc. No.:Q13729); and 21.49% identity and32.023% similarity with the Caenorhabditis elegans probable G proteincoupled receptor F01E11.5 (YDBM_CAEEL; Acc. No.:Q19084).

[0099] Variants of the HGPRBMY8 polypeptide are also encompassed by thepresent invention. A preferred HGPRBMY8 variant has at least 75 to 80%,more preferably at least 85 to 90%, and even more preferably at least90% amino acid sequence identity to the amino acid sequence claimedherein, and which retains at least one biological, immunological, orother functional characteristic or activity of the HGPRBMY8 polypeptide.Most preferred is a variant having at least 95% amino acid sequenceidentity to that of SEQ ID NO:2. For example, FIGS. 9 and 10 showmultiple sequence alignments of HGPRBMY8 and single nucleotidepolymorphism (SNP) variants. Highlighted are the differences insequence.

[0100] In a preferred embodiment, polynucleotide and polypeptidepolymorphisms are shown in FIGS. 18A-B. The standard one-letterabbreviation for amino acids is used to illustrate the deduced aminoacid sequence. The polynucleotide sequence contains a sequence of 1527nucleotides (SEQ ID NO:48), encoding a polypeptide of 508 amino acids(SEQ ID NO:49). The polynucleotide polymorphic sites are represented byan “N”, in bold. The polypeptide polymorphic sites are represented by an“X”, and underlined. The present invention encompasses thepolynucleotide at nucleotide position 370 as being either a “T” or a“G”, the polynucleotide at nucleotide position 1055 as being either a“A” or a “G”, the polynucleotide at nucleotide position 1192 as beingeither a “G” or a “A”, the polynucleotide at nucleotide position 1193 asbeing either a “C” or a “A”, and the polynucleotide at nucleotideposition 1194 as being either a “T” or a “G” of FIGS. 18A-B (SEQ IDNO:48), in addition to any combination thereof. The present inventionalso encompasses the polypeptide at amino acid position 124 as beingeither a “Leu” or a “Val”, the polypeptide at amino acid position 352 asbeing either a “Asp” or a “Gly”, and the polypeptide at amino acidposition 398 as being either a “Ala” or an “Lys” of FIGS. 18A-B (SEQ IDNO:49).

[0101] These polymorphisms are useful as genetic markers for any studythat attempts to look for linkage between HGPRBMY8 and a disease ordisease state related to this polypeptide.

[0102] In preferred embodiments, the following single nucleotidepolymorphism polynucleotides are encompassed by the present invention:CACCATTGTCTTGGTGTCAGT (SEQ ID NO:50), CACCATTGTCGTGGTGTCAGT (SEQ IDNO:51), GGTGAAGATGACATGGAGTTT (SEQ ID NO:52), GGTGAAGATGGCATGGAGTTT (SEQID NO:53), GTGCAAAGCTGCTAAAGTGAT (SEQ ID NO:54), GTGCAAAGCTACTAAAGTGAT(SEQ ID NO:55), TGCAAAGCTGCTAAAGTGATC (SEQ ID NO:56),TGCAAAGCTGATAAAGTGATC (SEQ ID NO:57) GCAAAGCTGCTAAAGTGATCT (SEQ IDNO:58), and/or GCAAAGCTGCGAAAGTGATCT (SEQ ID NO:59)

[0103] Polypeptides encoded by these polynucleotides are also provided.

[0104] The predicted ‘T’ to ‘G’ polynucleotide polymorphism located atnucleic acid 370 of SEQ ID NO:1 is a missense mutation resulting in achange in an encoding amino acid from ‘L’ to ‘V’ at amino acid position124 of SEQ ID NO:2.

[0105] The predicted ‘A’ to ‘G’ polynucleotide polymorphism located atnucleic acid 1055 of SEQ ID NO:1 is a missense mutation resulting in achange in an encoding amino acid from ‘D’ to ‘G’ at amino acid position352 of SEQ ID NO:2.

[0106] The predicted ‘G’ to ‘A’ polynucleotide polymorphism located atnucleic acid 1192 of SEQ ID NO:1 is a missense mutation resulting in achange in an encoding amino acid from ‘A’ to ‘T’ at amino acid position398 of SEQ ID NO:2.

[0107] The predicted ‘C’ to ‘A’ polynucleotide polymorphism located atnucleic acid 1193 of SEQ ID NO:1 is a missense mutation resulting in achange in an encoding amino acid from ‘A’ to ‘D’ at amino acid position398 of SEQ ID NO:2.

[0108] The predicted ‘T’ to ‘G’ polynucleotide polymorphism located atnucleic acid 1194 of SEQ ID NO:1 is a silent mutation and does notresult in a change in amino acid.

[0109] However, taken together the predicted ‘G’ to ‘A’ polynucleotidepolymorphism located at nucleic acid 1192, the predicted ‘C’ to ‘A’polynucleotide polymorphism located at nucleic acid 1193, and thepredicted ‘T’ to ‘G’ polynucleotide polymorphism located at nucleic acid1194 of SEQ ID NO:1 represent a missense mutations resulting in a changein an encoding amino acid from ‘A’ to ‘K’ at amino acid position 398 ofSEQ ID NO:2.

[0110] The present invention relates to isolated nucleic acid moleculescomprising, or alternatively, consisting of all or a portion of thevariant allele of the human HGPRBMY8 G-protein coupled receptor gene(e.g., wherein reference or wildtype human HGPRBMY8 G-protein coupledreceptor gene is exemplified by SEQ ID NO:1). Preferred portions are atleast 10, preferably at least 20, preferably at least 40, preferably atleast 100, contiguous polynucleotides comprising anyone of the humanHGPRBMY8 G-protein coupled receptor gene alleles described herein andexemplified in FIGS. 10A-D.

[0111] In one embodiment, the invention relates to a method forpredicting the likelihood that an individual will have a disorderassociated with the reference allele at nucleotide position 370, 1055,1192, 1193, and/or 1194 of SEQ ID NO:1 (or diagnosing or aiding in thediagnosis of such a disorder) comprising the steps of obtaining a DNAsample from an individual to be assessed and determining the nucleotidepresent at position 370, 1055, 1192, 1193, and/or 1194 of SEQ ID NO:1.The presence of the variant allele at this position indicates that theindividual has a greater likelihood of having a disorder associatedtherewith than an individual having the reference allele at thatposition, or a greater likelihood of having more severe symptoms.

[0112] Conversely, the invention relates to a method for predicting thelikelihood that an individual will have a disorder associated with thevariant allele at nucleotide position 370, 1055, 1192, 1193, and/or 1194of SEQ ID NO:1 (or diagnosing or aiding in the diagnosis of such adisorder) comprising the steps of obtaining a DNA sample from anindividual to be assessed and determining the nucleotide present atposition 370, 1055, 1192, 1193, and/or 1194 of SEQ ID NO:1. The presenceof the variant allele at this position indicates that the individual hasa greater likelihood of having a disorder associated therewith than anindividual having the reference allele at that position, or a greaterlikelihood of having more severe symptoms.

[0113] The present invention further relates to isolated proteins orpolypeptides comprising, or alternatively, consisting of all or aportion of the encoded variant amino acid sequence of the human HGPRBMY8G-protein coupled receptor polypeptide (e.g., wherein reference orwildtype human HGPRBMY8 G-protein coupled receptor polypeptide isexemplified by SEQ ID NO:2). Preferred portions are at least 10,preferably at least 20, preferably at least 40, preferably at least 100,contiguous polypeptides and comprises any one of the amino acid variantalleles of the human HGPRBMY8 G-protein coupled receptor polypeptideexemplified in FIGS. 18A-B, or a portion of SEQ ID NO:49. Alternatively,preferred portions are at least 10, preferably at least 20, preferablyat least 40, preferably at least 100, contiguous polypeptides andcomprises any one of the amino acid reference alleles of the humanHGPRBMY8 G-protein coupled receptor protein exemplified in FIGS. 18A-B,or a portion of SEQ ID NO:49. The invention further relates to isolatednucleic acid molecules encoding such polypeptides or proteins, as wellas to antibodies that bind to such proteins or polypeptides.

[0114] In another embodiment, the present invention encompassespolynucleotides, which encode the HGPRBMY8 polypeptide. Accordingly, anynucleic acid sequence, which encodes the amino acid sequence of HGPRBMY8polypeptide, can be used to produce recombinant molecules that expressHGPRBMY8 protein. In a particular embodiment, the present inventionencompasses the HGPRBMY8 polynucleotide comprising the nucleic acidsequence of SEQ ID NO:1 as shown in FIG. 1. More particularly, thepresent invention provides the HGPRBMY8 clone. More particularly, thepresent invention provides the HGPRBMY8 clone, deposited at the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209 on Jan. 24, 2001 and under ATCC Accession No. PTA-2966according to the terms of the Budapest Treaty.

[0115] As will be appreciated by the skilled practitioner in the art,the degeneracy of the genetic code results in the production of a numberof nucleotide sequences encoding HGPRBMY8 polypeptide. Some of thesequences bear minimal homology to the nucleotide sequences of any knownand naturally occurring gene. Accordingly, the present inventioncontemplates each and every possible variation of nucleotide sequencethat could be made by selecting combinations based on possible codonchoices. These combinations are made in accordance with the standardtriplet genetic code as applied to the nucleotide sequence of naturallyoccurring HGPRBMY8, and all such variations are to be considered asbeing specifically disclosed.

[0116] Although nucleotide sequences which encode HGPRBMY8 polypeptideand its variants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring HGPRBMY8 polypeptide underappropriately selected conditions of stringency, it may be advantageousto produce nucleotide sequences encoding HGPRBMY8 polypeptide, or itsderivatives, which possess a substantially different codon usage. Codonsmay be selected to increase the rate at which expression of thepeptide/polypeptide occurs in a particular prokaryotic or eukaryotichost in accordance with the frequency with which particular codons areutilized by the host. Other reasons for substantially altering thenucleotide sequence encoding HGPRBMY8 polypeptide, and its derivatives,without altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0117] The present invention also encompasses production of DNAsequences, or portions thereof, which encode the HGPRBMY8 polypeptide,and its derivatives, entirely by synthetic chemistry. After production,the synthetic sequence may be inserted into any of the many availableexpression vectors and cell systems using reagents that are well knownand practiced by those in the art. Moreover, synthetic chemistry andother known techniques may be used to introduce mutations into asequence encoding HGPRBMY8 polypeptide, or any fragment thereof.

[0118] In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of HGPRBMY8. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 1524 of SEQ ID NO:1, and the polypeptide corresponding to aminoacids 2 thru 508 of SEQ ID NO:2. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

[0119] Also encompassed by the present invention are polynucleotidesequences that are capable of hybridizing to the claimed nucleotidesequence of HGPRBMY8, such as that shown in SEQ ID NO:1, under variousconditions of stringency. Hybridization conditions are typically basedon the melting temperature (T_(m)) of the nucleic acid binding complexor probe (see, G. M. Wahl and S. L. Berger, 1987; Methods Enzymol.,152:399-407 and A. R. Kimmel, 1987; Methods of Enzymol., 152:507-511),and may be used at a defined stringency. For example, included in thepresent invention are sequences capable of hybridizing under moderatelystringent conditions to the HGPRBMY8 sequence of SEQ ID NO:1 and othersequences which are degenerate to those which encode HGPRBMY8polypeptide (e.g., as a non-limiting example: prewashing solution of 2×SSC, 0.5% SDS, 1.0 mM EDTA, pH 8.0, and hybridization conditions of 50°C., 5× SSC, overnight.

[0120] The nucleic acid sequence encoding the HGPRBMY8 protein may beextended utilizing a partial nucleotide sequence and employing variousmethods known in the art to detect upstream sequences such as promotersand regulatory elements. For example, one method, which may be employed,is restriction-site PCR, which utilizes universal primers to retrieveunknown sequence adjacent to a known locus (G. Sarkar, 1993, PCR MethodsApplic., 2:318-322). In particular, genomic DNA is first amplified inthe presence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0121] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region or sequence (T. Triglia etal., 1988, Nucleic Acids Res., 16:8186). The primers may be designedusing OLIGO 4.06 Primer Analysis software (National Biosciences Inc.;Plymouth, Minn.), or another appropriate program, to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Themethod uses several restriction enzymes to generate a suitable fragmentin the known region of a gene. The fragment is then circularized byintramolecular ligation and used as a PCR template.

[0122] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome (YAC) DNA (M. Lagerstrom et al., 1991,PCR Methods Applic., 1:111-119). In this method, multiple restrictionenzyme digestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR. J. D. Parker et al. (1991; Nucleic Acids Res.,19:3055-3060) provide another method which may be used to retrieveunknown sequences. In addition, PCR, nested primers, and PROMOTERFINDERlibraries can be used to walk genomic DNA (Clontech, Palo Alto, Calif.).This process avoids the need to screen libraries and is useful infinding intron/exon junctions.

[0123] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, since they will contain moresequences, which contain the 5′ regions of genes. The use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0124] The embodiments of the present invention can be practiced usingmethods for DNA sequencing which are well known and generally availablein the art. The methods may employ such enzymes as the Klenow fragmentof DNA polymerase I, SEQUENASE (US Biochemical Corp.; Cleveland, Ohio),Taq polymerase (PE Biosystems; Gaithersburg, Md.), thermostable T7polymerase (Amersham Pharmacia Biotechnology; Piscataway, N.J.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Life Technologies(Rockville, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton; Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research; Watertown, Mass.) and the ABICatalyst and 373 and 377 DNA sequencers (PE Biosystems; Gaithersburg,Md.).

[0125] Commercially available capillary electrophoresis systems may beused to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PEBiosystems; Gaithersburg, Md.) and the entire process—from loading ofsamples to computer analysis and electronic data display—may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA, which might be present in limitedamounts in a particular sample.

[0126] In another embodiment of the present invention, polynucleotidesequences or fragments thereof which encode HGPRBMY8 polypeptide, orpeptides thereof, may be used in recombinant DNA molecules to direct theexpression of HGPRBMY8 polypeptide product, or fragments or functionalequivalents thereof, in appropriate host cells. Because of the inherentdegeneracy of the genetic code, other DNA sequences, which encodesubstantially the same or a functionally equivalent amino acid sequence,may be produced and these sequences may be used to clone and expressHGPRBMY8 protein.

[0127] As will be appreciated by those having skill in the art, it maybe advantageous to produce HGPRBMY8 polypeptide-encoding nucleotidesequences possessing non-naturally occurring codons. For example, codonspreferred by a particular prokaryotic or eukaryotic host can be selectedto increase the rate of protein expression or to produce a recombinantRNA transcript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0128] The nucleotide sequence of the present invention can beengineered using methods generally known in the art in order to alterHGPRBMY8 polypeptide-encoding sequences for a variety of reasons,including, but not limited to, alterations which modify the cloning,processing, and/or expression of the gene product. DNA shuffling byrandom fragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Forexample, site-directed mutagenesis may be used to insert new restrictionsites, alter glycosylation patterns, change codon preference, producesplice variants, or introduce mutations, and the like.

[0129] In another embodiment of the present invention, natural,modified, or recombinant nucleic acid sequences encoding HGPRBMY8polypeptide may be ligated to a heterologous sequence to encode a fusionprotein. For example, for screening peptide libraries for inhibitors ofHGPRBMY8 activity, it may be useful to encode a chimeric HGPRBMY8protein that can be recognized by a commercially available antibody. Afusion protein may also be engineered to contain a cleavage site locatedbetween the HGPRBMY8 protein-encoding sequence and the heterologousprotein sequence, so that HGPRBMY8 protein may be cleaved and purifiedaway from the heterologous moiety.

[0130] In another embodiment, sequences encoding HGPRBMY8 polypeptidemay be synthesized in whole, or in part, using chemical methods wellknown in the art (see, for example, M. H. Caruthers et al., 1980, Nucl.Acids Res. Symp. Ser., 215-223 and T. Horn et al., 1980, Nucl. AcidsRes. Symp. Ser., 225-232). Alternatively, the protein itself may beproduced using chemical methods to synthesize the amino acid sequence ofHGPRBMY8 polypeptide, or a fragment or portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(J. Y. Roberge et al., 1995, Science, 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (PE Biosystems; Gaithersburg, Md.).

[0131] The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., T. Creighton,1983, Proteins, Structures and Molecular Principles, W. H. Freeman andCo., New York, N.Y.), by reversed-phase high performance liquidchromatography, or other purification methods as are known in the art.The composition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure;Creighton, supra). In addition, the amino acid sequence of HGPRBMY8polypeptide or any portion thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0132] To express a biologically active HGPRBMY8 polypeptide or peptide,the nucleotide sequences encoding HGPRBMY8 polypeptide, or functionalequivalents, may be inserted into an appropriate expression vector,i.e., a vector, which contains the necessary elements for thetranscription and translation of the inserted coding sequence.

[0133] Methods, which are well known to those skilled in the art, may beused to construct expression vectors containing sequences encodingHGPRBMY8 polypeptide and appropriate transcriptional and translationalcontrol elements. These methods include in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Such techniques are described in J. Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.and in F. M. Ausubel et al., 1989, Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y.

[0134] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding HGPRBMY8 polypeptide. Suchexpression vector/host systems include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) and tobacco mosaic virus (TMV)), or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems. The host cell employed is not limiting to the presentinvention.

[0135] “Control elements” or “regulatory sequences” are thosenon-translated regions of the vector, e.g., enhancers, promoters, 5′ and3′ untranslated regions, which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene; LaJolla, Calif.) or PSPORT1 plasmid (Life Technologies; Rockville, Md.),and the like, may be used. The baculovirus polyhedrin promoter may beused in insect cells. Promoters or enhancers derived from the genomes ofplant cells (e.g., heat shock, RUBISCO; and storage protein genes), orfrom plant viruses (e.g., viral promoters or leader sequences), may becloned into the vector. In mammalian cell systems, promoters frommammalian genes or from mammalian viruses are preferred. If it isnecessary to generate a cell line that contains multiple copies of thesequence encoding HGPRBMY8, vectors based on SV40 or EBV may be usedwith an appropriate selectable marker.

[0136] In bacterial systems, a number of expression vectors may beselected, depending upon the use intended for the expressed HGPRBMY8product. For example, when large quantities of expressed protein areneeded for the induction of antibodies, vectors, which direct high levelexpression of fusion proteins that are readily purified, may be used.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as BLUESCRIPT (Stratagene; LaJolla, Calif.), in which the sequence encoding HGPRBMY8 polypeptide maybe ligated into the vector in-frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase, sothat a hybrid protein is produced; pIN vectors (see, G. Van Heeke and S.M. Schuster, 1989, J. Biol. Chem., 264:5503-5509); and the like. pGEXvectors (Promega, Madison, Wis.) may also be used to express foreignpolypeptides, as fusion proteins with glutathione S-transferase (GST).In general, such fusion proteins are soluble and can be easily purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0137] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. (For reviews, see F. M. Ausubel etal., supra, and Grant et al., 1987, Methods Enzymol., 153:516-544).

[0138] Should plant expression vectors be desired and used, theexpression of sequences encoding HGPRBMY8 polypeptide may be driven byany of a number of promoters. For example, viral promoters such as the35S and 19S promoters of CaMV may be used alone or in combination withthe omega leader sequence from TMV (N. Takamatsu, 1987, EMBO J.,6:307-311). Alternatively, plant promoters such as the small subunit ofRUBISCO, or heat shock promoters, may be used (G. Coruzzi et al., 1984,EMBO J., 3:1671-1680; R. Broglie et al., 1984, Science, 224:838-843; andJ. Winter et al., 1991, Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,S. Hobbs or L. E. Murry, In: McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0139] An insect system may also be used to express HGPRBMY8polypeptide. For example, in one such system, Autographa californicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.The sequences encoding HGPRBMY8 polypeptide may be cloned into anon-essential region of the virus such as the polyhedrin gene and placedunder control of the polyhedrin promoter. Successful insertion ofHGPRBMY8 polypeptide will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which the HGPRBMY8 polypeptide product may beexpressed (E. K. Engelhard et al., 1994, Proc. Nat. Acad. Sci.,91:3224-3227).

[0140] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding HGPRBMY8 polypeptide may beligated into an adenovirus transcription/translation complex containingthe late promoter and tripartite leader sequence. Insertion in anon-essential E1 or E3 region of the viral genome may be used to obtaina viable virus which is capable of expressing HGPRBMY8 polypeptide ininfected host cells (J. Logan and T. Shenk, 1984, Proc. Natl. Acad.Sci., 81:3655-3659). In addition, transcription enhancers, such as theRous sarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

[0141] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding HGPRBMY8 polypeptide. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding HGPRBMY8 polypeptide, its initiationcodon, and upstream sequences are inserted into the appropriateexpression vector, no additional transcriptional or translationalcontrol signals may be needed. However, in cases where only codingsequence, or a fragment thereof, is inserted, exogenous translationalcontrol signals, including the ATG initiation codon, should be provided.Furthermore, the initiation codon should be in the correct reading frameto ensure translation of the entire insert. Exogenous translationalelements and initiation codons may be of various origins, both naturaland synthetic. The efficiency of expression may be enhanced by theinclusion of enhancers which are appropriate for the particular cellsystem that is used, such as those described in the literature (D.Scharf et al., 1994, Results Probl. Cell Differ., 20:125-162).

[0142] Moreover, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells having specific cellular machinery andcharacteristic mechanisms for such post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138) are available from the American TypeCulture Collection (ATCC), American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209, and may be chosento ensure the correct modification and processing of the foreignprotein.

[0143] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress HGPRBMY8 protein may be transformed using expression vectorswhich may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same, or on aseparate, vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched cell culture medium beforethey are switched to selective medium. The purpose of the selectablemarker is to confer resistance to selection, and its presence allows thegrowth and recovery of cells, which successfully express the introducedsequences. Resistant clones of stably transformed cells may beproliferated using tissue culture techniques appropriate to the celltype.

[0144] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theHerpes Simplex Virus thymidine kinase (HSV TK), (M. Wigler et al., 1977,Cell, 11:223-32) and adenine phosphoribosyltransferase (I. Lowy et al.,1980, Cell, 22:817-23) genes which can be employed in tk⁻ or aprt⁻cells, respectively. Also, anti-metabolite, antibiotic or herbicideresistance can be used as the basis for selection; for example, dhfr,which confers resistance to methotrexate (M. Wigler et al., 1980, Proc.Natl. Acad. Sci., 77:3567-70); npt, which confers resistance to theaminoglycosides neomycin and G-418 (F. Colbere-Garapin et al., 1981, J.Mol. Biol., 150:1-14); and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively(Murry, supra). Additional selectable genes have been described, forexample, trpB, which allows cells to utilize indole in place oftryptophan, or hisD, which allows cells to utilize histinol in place ofhistidine (S. C. Hartman and R. C. Mulligan, 1988, Proc. Natl. Acad.Sci., 85:8047-51). Recently, the use of visible markers has gainedpopularity with such markers as the anthocyanins, β-glucuronidase andits substrate GUS, and luciferase and its substrate luciferin, which arewidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression that is attributable toa specific vector system (C. A. Rhodes et al., 1995, Methods Mol. Biol.,55:121-131).

[0145] Although the presence or absence of marker gene expressionsuggests that the gene of interest is also present, the presence andexpression of the desired gene of interest may need to be confirmed. Forexample, if the nucleic acid sequence encoding HGPRBMY8 polypeptide isinserted within a marker gene sequence, recombinant cells containingsequences encoding HGPRBMY8 polypeptide can be identified by the absenceof marker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HGPRBMY8 polypeptide under the controlof a single promoter. Expression of the marker gene in response toinduction or selection usually indicates co-expression of the tandemgene.

[0146] Alternatively, host cells, which contain the nucleic acid,sequence encoding HGPRBMY8 polypeptide and which express HGPRBMY8polypeptide product may be identified by a variety of procedures knownto those having skill in the art. These procedures include, but are notlimited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques, including membrane, solution, or chip basedtechnologies, for the detection and/or quantification of nucleic acid orprotein.

[0147] The presence of polynucleotide sequences encoding HGPRBMY8polypeptide can be detected by DNA-DNA or DNA-RNA hybridization, or byamplification using probes or portions or fragments of polynucleotidesencoding HGPRBMY8 polypeptide. Nucleic acid amplification based assaysinvolve the use of oligonucleotides or oligomers, based on the sequencesencoding HGPRBMY8 polypeptide, to detect transformants containing DNA orRNA encoding HGPRBMY8 polypeptide.

[0148] A wide variety of labels and conjugation techniques are known andemployed by those skilled in the art and may be used in various nucleicacid and amino acid assays. Means for producing labeled hybridization orPCR probes for detecting sequences related to polynucleotides encodingHGPRBMY8 polypeptide include oligo-labeling, nick translation,end-labeling, or PCR amplification using a labeled nucleotide.Alternatively, the sequences encoding HGPRBMY8 polypeptide, or anyportions or fragments thereof, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase, such as T7, T3, orSP(6) and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (e.g., Amersham PharmaciaBiotech, Promega and U.S. Biochemical Corp.). Suitable reportermolecules or labels which may be used include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

[0149] Furthermore, in yet another embodiment, G-protein coupledreceptor-encoding polynucleotide sequences can be used to purify amolecule or compound in a sample, wherein the molecule or compoundspecifically binds to the polynucleotide, comprising: a) combining theG-protein coupled receptor-encoding polynucleotide, or fragment thereof,under conditions to allow specific binding; b) detecting specificbinding between the G-protein coupled receptor-encoding polynucleotideand the molecule or compound; c) recovering the bound polynucleotide;and d) separating the polynucleotide from the molecule or compound,thereby obtaining a purified molecule or compound.

[0150] Host cells transformed with nucleotide sequences encodingHGPRBMY8 protein, or fragments thereof, may be cultured under conditionssuitable for the expression and recovery of the protein from cellculture. The protein produced by a recombinant cell may be secreted orcontained intracellularly depending on the sequence and/or the vectorused. As will be understood by those having skill in the art, expressionvectors containing polynucleotides which encode HGPRBMY8 protein may bedesigned to contain signal sequences which direct secretion of theHGPRBMY8 protein through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join nucleic acid sequences encodingHGPRBMY8 protein to nucleotide sequence encoding a polypeptide domain,which will facilitate purification of soluble proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals; protein A domains that allowpurification on immobilized immunoglobulin; and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and HGPRBMY8 protein may be usedto facilitate purification. One such expression vector provides forexpression of a fusion protein containing HGPRBMY8 and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMAC(immobilized metal ion affinity chromatography) as described by J.Porath et al., 1992, Prot. Exp. Purif., 3:263-281, while theenterokinase cleavage site provides a means for purifying from thefusion protein. For a discussion of suitable vectors for fusion proteinproduction, see D. J. Kroll et al., 1993; DNA Cell Biol., 12:441-453.

[0151] In addition to recombinant production, fragments of HGPRBMY8polypeptide may be produced by direct peptide synthesis usingsolid-phase techniques (J. Merrifield, 1963, J. Am. Chem. Soc.,85:2149-2154). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using ABI 431A Peptide Synthesizer (PE Biosystems;Gaithersburg, Md.). Various fragments of HGPRBMY8 polypeptide can bechemically synthesized separately and then combined using chemicalmethods to produce the full-length molecule.

[0152] Human artificial chromosomes (HACs) may be used to deliver largerfragments of DNA than can be contained and expressed in a plasmidvector. HACs are linear microchromosomes which may contain DNA sequencesof 10K to 10M in size, and contain all of the elements that are requiredfor stable mitotic chromosome segregation and maintenance (see, J. J.Harrington et al., 1997, Nature Genet., 15:345-355). HACs of 6 to 10Mare constructed and delivered via conventional delivery methods (e.g.,liposomes, polycationic amino polymers, or vesicles) for therapeuticpurposes.

Diagnostic Assays

[0153] A variety of protocols for detecting and measuring the expressionof HGPRBMY8 polypeptide using either polyclonal or monoclonal antibodiesspecific for the protein are known and practiced in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactivewith two non-interfering epitopes on the HGPRBMY8 polypeptide ispreferred, but a competitive binding assay may also be employed. Theseand other assays are described in the art as represented by thepublication of R. Hampton et al., 1990; Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn. and D. E. Maddox et al.,1983; J. Exp. Med., 158:1211-1216).

[0154] This invention also relates to the use of HGPRBMY8polynucleotides as diagnostic reagents. Detection of a mutated form ofthe HGPRBMY8 gene associated with a dysfunction provides a diagnostictool that can add to or define a diagnosis of a disease orsusceptibility to a disease which results from under-expression,over-expression, or altered expression of HGPRBMY8. Individuals carryingmutations in the HGPRBMY8 gene may be detected at the DNA level by avariety of techniques.

[0155] Nucleic acids for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniquesprior to analysis. RNA or cDNA may also be used in similar fashion.Deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Hybridizingamplified DNA to labeled HGPRBMY8 polynucleotide sequences can identifypoint mutations. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing. See, e.g.,Myers et al., Science (1985) 230:1242. Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method. See Cotton etal., Proc. Natl. Acad. Sci., USA (1985) 85:43297-4401. In anotherembodiment, an array of oligonucleotides probes comprising HGPRBMY8nucleotide sequence or fragments thereof can be constructed to conductefficient screening of e.g., genetic mutations. Array technology methodsare well known and have general applicability and can be used to addressa variety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability (see for example: M. Chee etal., Science, 274:610-613, 1996).

[0156] The diagnostic assays offer a process for diagnosing ordetermining, for example, a susceptibility to infections such asbacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2 through detection of a mutation inthe HGPRBMY8 gene by the methods described. The invention also providesdiagnostic assays for determining or monitoring susceptibility to thefollowing conditions, diseases, or disorders: HIV infections; asthma;allergies; obesity; anorexia; bulimia; ulcers; acute heart failure;hypotension; hypertension; angina pectoris; myocardial infarction;urinary retention; osteoporosis; benign prostatic hypertrophy; cancers;brain-related disorders; Parkinson's disease; neuropathic pain; immune;metabolic; cardiovascular; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome; Sydenham chorea; major depressivedisorder; and obsessive-compulsive disorder (OCD). Movement typediseases, disorders, or conditions may be targeted in particular sinceHGPRBMY8 is expressed in the caudate nucleus of the brain.

[0157] In addition, infections such as bacterial, fungal, protozoan andviral infections, particularly infections caused by HIV-1 or HIV-2, aswell as, conditions, diseases, or disorders such as, HIV infections;asthma; allergies; obesity; anorexia; bulimia; ulcers; acute heartfailure; hypotension; hypertension; angina pectoris; myocardialinfarction; urinary retention; osteoporosis; benign prostatichypertrophy; cancers; brain-related disorders; Parkinson's disease;neuropathic pain; immune; metabolic; cardiovascular; and psychotic andneurological disorders, including anxiety, schizophrenia, manicdepression, delirium, dementia, severe mental retardation anddyskinesias, such as Huntington's disease or Gilles dela Tourett'ssyndrome, can be diagnosed by methods comprising determining from asample derived from a subject having an abnormally decreased orincreased level of HGPRBMY8 polypeptide or HGPRBMY8 mRNA. Decreased orincreased expression can be measured at the RNA level using any of themethods well known in the art for the quantification of polynucleotides,such as, for example, PCR, RT-PCR, RNAse protection, Northern blottingand other hybridization methods. Assay techniques that can be used todetermine levels of a protein, such as an HGPRBMY8, in a sample derivedfrom a host are well known to those of skill in the art. Such assaymethods include radioimmunoassays, competitive-binding assays, WesternBlot analysis and ELISA assays.

[0158] In another of its aspects, the present invention relates to adiagnostic kit for a disease or susceptibility to a disease,particularly infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2, as wellas, conditions, diseases, or disorders such as, HIV infections; asthma;allergies; obesity; anorexia; bulimia; ulcers; acute heart failure;hypotension; hypertension; angina pectoris; myocardial infarction;urinary retention; osteoporosis; benign prostatic hypertrophy; cancers;brain-related disorders; Parkinson's disease; neuropathic pain; immune;metabolic; cardiovascular; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Gilles dela Tourett's syndrome, which comprises:

[0159] (a) an HGPRBMY8 polynucleotide, preferably the nucleotidesequence of SEQ ID NO:1, or a fragment thereof; or

[0160] (b) a nucleotide sequence complementary to that of (a); or

[0161] (c) an HGPRBMY8 polypeptide, preferably the polypeptide of SEQ IDNO:2, or a fragment thereof; or

[0162] (d) an antibody to an HGPRBMY8 polypeptide, preferably to thepolypeptide of SEQ ID NO:2, or combinations thereof.

[0163] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component and instructions are frequentlyincluded.

[0164] The GPCR polynucleotides which may be used in the diagnosticassays according to the present invention include oligonucleotidesequences, complementary RNA and DNA molecules, and PNAs. Thepolynucleotides may be used to detect and quantify HGPRBMY8-encodingnucleic acid expression in biopsied tissues in which expression (orunder- or overexpression) of the HGPRBMY8 polynucleotide may becorrelated with disease. The diagnostic assays may be used todistinguish between the absence, presence, and excess expression ofHGPRBMY8, and to monitor regulation of HGPRBMY8 polynucleotide levelsduring therapeutic treatment or intervention.

[0165] In a related aspect, hybridization with PCR probes which arecapable of detecting polynucleotide sequences, including genomicsequences, encoding HGPRBMY8 polypeptide, or closely related molecules,may be used to identify nucleic acid sequences which encode HGPRBMY8polypeptide. The specificity of the probe, whether it is made from ahighly specific region, e.g., about 8 to 10 contiguous nucleotides inthe 5′ regulatory region, or a less specific region, e.g., especially inthe 3′ coding region, and the stringency of the hybridization oramplification (maximal, high, intermediate, or low) will determinewhether the probe identifies only naturally occurring sequences encodingHGPRBMY8 polypeptide, alleles thereof, or related sequences.

[0166] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides, mostoptimally 15-35 nucleotides, encoding the HGPRBMY8 polypeptide. Thehybridization probes of this invention may be DNA or RNA and may bederived from the nucleotide sequence of SEQ ID NO:1, or from genomicsequence including promoter, enhancer elements, and introns of thenaturally occurring HGPRBMY8 protein.

[0167] Methods for producing specific hybridization probes for DNAencoding the HGPRBMY8 polypeptide include the cloning of a nucleic acidsequence that encodes the HGPRBMY8 polypeptide, or HGPRBMY8 derivatives,into vectors for the production of mRNA probes. Such vectors are knownin the art, commercially available, and may be used to synthesize RNAprobes in vitro by means of the addition of the appropriate RNApolymerases and the appropriate labeled nucleotides. Hybridizationprobes may be labeled by a variety of detector/reporter groups, e.g.,radionuclides such as ³²P or ³⁵S, or enzymatic labels, such as alkalinephosphatase coupled to the probe via avidin/biotin coupling systems, andthe like.

[0168] The polynucleotide sequence encoding the HGPRBMY8 polypeptide, orfragments thereof, may be used for the diagnosis of disorders associatedwith expression of HGPRBMY8. Examples of such disorders or conditionsare described for “Therapeutics”. The polynucleotide sequence encodingthe HGPRBMY8 polypeptide may be used in Southern or Northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; orin dip stick, pin, ELISA or chip assays utilizing fluids or tissues frompatient biopsies to detect the status of, e.g., levels or overexpressionof HGPRBMY8, or to detect altered HGPRBMY8 expression. Such qualitativeor quantitative methods are well known in the art.

[0169] In a particular aspect, the nucleotide sequence encoding theHGPRBMY8 polypeptide may be useful in assays that detect activation orinduction of various neoplasms or cancers, particularly those mentionedsupra. The nucleotide sequence encoding the HGPRBMY8 polypeptide may belabeled by standard methods, and added to a fluid or tissue sample froma patient, under conditions suitable for the formation of hybridizationcomplexes. After a suitable incubation period, the sample is washed andthe signal is quantified and compared with a standard value. If theamount of signal in the biopsied or extracted sample is significantlyaltered from that of a comparable control sample, the nucleotidesequence has hybridized with nucleotide sequence present in the sample,and the presence of altered levels of nucleotide sequence encoding theHGPRBMY8 polypeptide in the sample indicates the presence of theassociated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0170] To provide a basis for the diagnosis of disease associated withexpression of HGPRBMY8, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes the HGPRBMY8 polypeptide,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject (patient) values is used to establish the presenceof disease.

[0171] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in a normal individual. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0172] With respect to cancer, the presence of an abnormal amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier, thereby preventing the development or furtherprogression of the cancer.

[0173] Additional diagnostic uses for oligonucleotides designed from thenucleic acid sequence encoding the HGPRBMY8 polypeptide may involve theuse of PCR. Such oligomers may be chemically synthesized, generatedenzymatically, or produced from a recombinant source. Oligomers willpreferably comprise two nucleotide sequences, one with sense orientation(5′→3′) and another with antisense (3′→5′), employed under optimizedconditions for identification of a specific gene or condition. The sametwo oligomers, nested sets of oligomers, or even a degenerate pool ofoligomers may be employed under less stringent conditions for detectionand/or quantification of closely related DNA or RNA sequences.

[0174] Methods suitable for quantifying the expression of HGPRBMY8include radiolabeling or biotinylating nucleotides, co-amplification ofa control nucleic acid, and standard curves onto which the experimentalresults are interpolated (P. C. Melby et al., 1993, J. Immunol. Methods,159:235-244; and C. Duplaa et al., 1993, Anal. Biochem., 229-236). Thespeed of quantifying multiple samples may be accelerated by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantification.

Therapeutic Assays

[0175] The HGPRBMY8 polypeptide (SEQ ID NO:2) shares homology withsomatostatin-type receptors. The HGPRBMY8 protein may play a role inneurological disorders, and/or in cell cycle regulation, and/or in cellsignaling. The HGPRBMY8 protein may further be involved in neoplastic,cardiovascular, and immunological disorders.

[0176] In one embodiment of the present invention, the HGPRBMY8 proteinmay play a role in neoplastic disorders. An antagonist or inhibitor ofthe HGPRBMY8 polypeptide may be administered to an individual to preventor treat a neoplastic disorder. Such disorders may include, but are notlimited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, and teratocarcinoma, and particularly, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In a related aspect, anantibody which specifically binds to HGPRBMY8 may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express theHGPRBMY8 polypeptide.

[0177] In another embodiment of the present invention, an antagonist orinhibitory agent of the HGPRBMY8 polypeptide may be administered to anindividual to prevent or treat an immunological disorder. Such disordersmay include, but are not limited to, AIDS, Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma.

[0178] In a preferred embodiment of the present invention, an antagonistor inhibitory agent of the HGPRBMY8 polypeptide may be administered toan individual to prevent or treat a neurological disorder, particularlysince HGPRBMY8 is highly expressed in the brain. Such disorders mayinclude, but are not limited to, akathesia, Alzheimer's disease,amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia,cerebral neoplasms, dementia, depression, Down's syndrome, tardivedyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, Parkinson's disease, paranoid psychoses, schizophrenia, andTourette's disorder.

[0179] In preferred embodiments, the HGPRBMY8 polynucleotides andpolypeptides, including agonists, antagonists, and fragments thereof,are useful for modulating intracellular cAMP associated signalingpathways.

[0180] In another embodiment of the present invention, an expressionvector containing the complement of the polynucleotide encoding HGPRBMY8polypeptide may be administered to an individual to treat or prevent aneoplastic disorder, including, but not limited to, the types of cancersand tumors described above.

[0181] In yet another embodiment of the present invention, an expressionvector containing the complement of the polynucleotide encoding HGPRBMY8polypeptide may be administered to an individual to treat or prevent animmune disorder, including, but not limited to, the types of immunedisorders described above.

[0182] In a preferred embodiment of the present invention, an expressionvector containing the complement of the polynucleotide encoding HGPRBMY8polypeptide may be administered to an individual to treat or prevent aneurological disorder, including, but not limited to, the types ofdisorders described above.

[0183] In another embodiment, the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the present inventioncan be administered in combination with other appropriate therapeuticagents. Selection of the appropriate agents for use in combinationtherapy may be made by one of ordinary skill in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

[0184] Antagonists or inhibitors of the HGPRBMY8 polypeptide of thepresent invention may be produced using methods which are generallyknown in the art. For example, the HGPRBMY8 transfected CHO-NFAT/CREcell lines of the present invention are useful for the identification ofagonists and antagonists of the HGPRBMY8 polypeptide. Representativeuses of these cell lines would be their inclusion in a method ofidentifying HGPRBMY8 agonists and antagonists. Preferably, the celllines are useful in a method for identifying a compound that modulatesthe biological activity of the HGPRBMY8 polypeptide, comprising thesteps of (a) combining a candidate modulator compound with a host cellexpressing the HGPRBMY8 polypeptide having the sequence as set forth inSEQ ID NO:2; and (b) measuring an effect of the candidate modulatorcompound on the activity of the expressed HGPRBMY8 polypeptide.Representative vectors expressing the HGPRBMY8 polypeptide arereferenced herein (e.g., pcDNA3.1 hygro™) or otherwise known in the art.

[0185] The cell lines are also useful in a method of screening for acompounds that is capable of modulating the biological activity ofHGPRBMY8 polypeptide, comprising the steps of: (a) determining thebiological activity of the HGPRBMY8 polypeptide in the absence of amodulator compound; (b) contacting a host cell expression the HGPRBMY8polypeptide with the modulator compound; and (c) determining thebiological activity of the HGPRBMY8 polypeptide in the presence of themodulator compound; wherein a difference between the activity of theHGPRBMY8 polypeptide in the presence of the modulator compound and inthe absence of the modulator compound indicates a modulating effect ofthe compound. Additional uses for these cell lines are described hereinor otherwise known in the art. In particular, purified HGPRBMY8 protein,or fragments thereof, can be used to produce antibodies, or to screenlibraries of pharmaceutical agents, to identify those which specificallybind HGPRBMY8.

[0186] Antibodies specific for HGPRBMY8 polypeptide, or immunogenicpeptide fragments thereof, can be generated using methods that have longbeen known and conventionally practiced in the art. Such antibodies mayinclude, but are not limited to, polyclonal, monoclonal, chimeric,single chain, Fab fragments, and fragments produced by an Fab expressionlibrary. Neutralizing antibodies, (i.e., those which inhibit dimerformation) are especially preferred for therapeutic use.

[0187] The present invention also encompasses the polypeptide sequencesthat intervene between each of the predicted HGPRBMY8 transmembranedomains. Since these regions are solvent accessible eitherextracellularly or intracellularly, they are particularly useful fordesigning antibodies specific to each region. Such antibodies may beuseful as antagonists or agonists of the HGPRBMY8 full-lengthpolypeptide and may modulate its activity.

[0188] The following serve as non-limiting examples of peptides orfragments that may be used to generate antibodies:MTSTCTNSTRESNSSHTCMPLSKMPISLAHGIIRST (SEQ ID NO:26) QRKPQLLQVTNRF (SEQID NO:27) WPLNS (SEQ ID NO:28) DRYLSIIHPLSYPSKMTQRR (SEQ ID NO:29)GQAAFDERNALCSMIWGASPSYT (SEQ ID NO:30)CAARRQHALLYNVKRHSLEVRVKDCVENEDEEGAEKKEEFQDESEFRRQ (SEQ ID NO:31)HEGEVKAKEGRMEAKDGSLKAKEGSTGTSESSVEAGSEEVRESSTVASDGSMEGKEGSTKVEENSMKADKGRTEVNQCSIDLGEDDMEFGEDDINFSEDDVEAVNIPESLPPSRRNSNSNPPLPRCYQCKAAK AVLAVWVDVETQVPQ (SEQ ID NO:32)YGYMHKTIKKEIQDMLKKFFCKEKPPKEDSHPDLPGTEGGTEGKIVPSYD (SEQ ID NO:33) SATFP

[0189] The present invention also encompasses the polypeptide sequencesthat intervene between each of the predicted HGPRBMY8 transmembranedomains. Since these regions are solvent accessible eitherextracellularly or intracellularly, they are particularly useful fordesigning antibodies specific to each region. Such antibodies may beuseful as antagonists or agonists of the HGPRBMY8 full-lengthpolypeptide and may modulate its activity.

[0190] In preferred embodiments, the following N-terminal HGPRBMY8 TM1-2intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: Q1-F13, R2-F13, K3-F13, P4-F13, Q5-F13, L6-F13,and/or L7-F13 of SEQ ID NO:27. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal HGPRBMY8 TM1-2 intertransmembrane domaindeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0191] In preferred embodiments, the following C-terminal HGPRBMY8 TM1-2intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: Q1-F13, Q1-R12, Q1-11, Q1-T10, Q1-V9, Q1-Q8, and/orQ1-L7 of SEQ ID NO:27. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal HGPRBMY8 TM1-2 intertransmembrane domaindeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0192] In preferred embodiments, the following N-terminal HGPRBMY8 TM3-4intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: D1-R20, R2-R20, Y3-R20, L4-R20, S5-R20, I6-R20,I7-R20, H8-R20, P9-R20, L10-R20, S11-R20, Y12-R20, P13-R20, and/orS14-R20 of SEQ ID NO:29. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal HGPRBMY8 TM3-4 intertransmembrane domaindeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0193] In preferred embodiments, the following C-terminal HGPRBMY8 TM3-4intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: D1-R20, D1-R19, D1-Q18, D1-T17, D1-M16, D1-K15,D1-S14, D1-P13, D1-Y12, D1-S11, D1-L10, D1-P9, D1-H8, and/or D1-I7 ofSEQ ID NO:29. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseC-terminal HGPRBMY8 TM3-4 intertransmembrane domain deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0194] In preferred embodiments, the following N-terminal HGPRBMY8 TM4-5intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: G1-T23, Q2-T23, A3-T23, A4-T23, F5-T23, D6-T23,E7-T23, R8-T23, N9-T23, A10-T23, L11-T23, C12-T23, S13-T23, M14-T23,I15-T23, W16-T23, and/or G17-T23 of SEQ ID NO:30. Polynucleotidesequences encoding these polypeptides are also provided. The presentinvention also encompasses the use of these N-terminal HGPRBMY8 TM4-5intertransmembrane domain deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0195] In preferred embodiments, the following C-terminal HGPRBMY8 TM4-5intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: G1-T23, G1-Y22, G1-S21, G1-P20, G1-S19, G1-A18,G1-G17, G1-W16, G1-I15, G1-M14, G1-S13, G1-C12, G1-L11, G1-A10, G1-N9,G1-R8, and/or G1-E7 of SEQ ID NO:30. Polynucleotide sequences encodingthese polypeptides are also provided. The present invention alsoencompasses the use of these C-terminal HGPRBMY8 TM4-5intertransmembrane domain deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0196] In preferred embodiments, the following N-terminal HGPRBMY8 TM5-6intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: C1-K182, A2-K182, A3-K182, R4-K182, R5-K182, Q6-K182,H7-K182, A8-K182, L9-K182, L10-K182, Y11-K182, N12-K182, V13-K182,K14-K182, R15-K182, H16-K182, S17-K182, L18-K182, E19-K182, V20-K182,R21-K182, V22-K182, K23-K182, D24-K182, C25-K182, V26-K182, E27-K182,N28-K182, E29-K182, D30-K182, E31-K182, E32-K182, G33-K182, A34-K182,E35-K182, K36-K182, K37-K182, E38-K182, E39-K182, F40-K182, Q41-K182,D42-K182, E43-K182, S44-K182, E45-K182, F46-K182, R47-K182, R48-K182,Q49-K182, H50-K182, E51-K182, G52-K182, E53-K182, V54-K182, K55-K182,A56-K182, K57-K182, E58-K182, G59-K182, R60-K182, M61-K182, E62-K182,A63-K182, K64-K182, D65-K182, G66-K182, S67-K182, L68-K182, K69-K182,A70-K182, K71-K182, E72-K182, G73-K182, S74-K182, T75-K182, G76-K182,T77-K182, S78-K182, E79-K182, S80-K182, S81-K182, V82-K182, E83-K182,A84-K182, G85-K182, S86-K182, E87-K182, E88-K182, V89-K182, R90-K182,E91-K182, S92-K182, S93-K182, T94-K182, V95-K182, A96-K182, S97-K182,D98-K182, G99-K182, S100-K182, M101-K182, E102-K182, G103-K182,K104-K182, E105-K182, G106-K182, S107-K182, T108-K182, K109-K182,V110-K182, E111-K182, E112-K182, N113-K182, S114-K182, M115-K182,K116-K182, A117-K182, D118-K182, K119-K182, G120-K182, R121-K182,T122-K182, E123-K182, V124-K182, N125-K182, Q126-K182, C127-K182,S128-K182, I129-K182, D130-K182, L131-K182, G132-K182, E133-K182,D134-K182, D135-K182, M136-K182, E137-K182, F138-K182, G139-K182,E140-K182, D141-K182, D142-K182, I143-K182, N144-K182, F145-K182,S146-K182, E147-K182, D148-K182, D149-K182, V150-K182, E151-K182,A152-K182, V153-K182, N154-K182, I155-K182, P156-K182, E157-K182,S158-K182, L159-K182, P160-K182, P161-K182, S162-K182, R163-K182,R164-K182, N165-K182, S166-K182, N167-K182, S168-K182, N169-K182,P170-K182, P171-K182, L172-K182, P173-K182, R174-K182, C175-K182, and/orY176-K182 of SEQ ID NO:31. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal HGPRBMY8 TM5-6 intertransmembrane domaindeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0197] In preferred embodiments, the following C-terminal HGPRBMY8 TM5-6intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: C1-K182, C1-A181, C1-A180, C1-K179, C1-C178, C1-Q177,C1-Y176, C1-C175, C1-R174, C1-P173, C1-L172, C1-P171, C1-P170, C1-N169,C1-S168, C1-N167, C1-S166, C1-N165, C1-R164, C1-R163, C1-S162, C1-P161,C1-P160, C1-L159, C1-S158, C1-E157, C1-P156, C1-I155, C1-N154, C1-V153,C1-A152, C1-E151, C1-V150, C1-D149, C1-D148, C1-E147, C1-S146, C1-F145,C1-N144, C1-I143, C1-D142, C1-D141, C1-E140, C1-G139, C1-F138, C1-E137,C1-M136, C1-D135, C1-D134, C1-E133, C1-G132, C1-L131, C1-D130, C1-I129,C1-S128, C1-C127, C1-Q126, C1-N125, C1-V124, C1-E123, C1-T122, C1-R121,C1-G120, C1-K119, C1-D118, C1-A117, C1-K116, C1-M115, C1-S114, C1-N113,C1-E112, C1-E111, C1-V110, C1-K109, C1-T108, C1-S107, C1-G106, C1-E105,C1-K104, C1-G103, C1-E102, C1-M101, C1-S100, C1-G99, C1-D98, C1-S97,C1-A96, C1-V95, C1-T94, C1-S93, C1-S92, C1-E91, C1-R90, C1-V89, C1-E88,C1-E87, C1-S86, C1-G85, C1-A84, C1-E83, C1-V82, C1-S81, C1-S80, C1-E79,C1-S78, C1-T77, C1-G76, C1-T75, C1-S74, C1-G73, C1-E72, C1-K71, C1-A70,C1-K69, C1-L68, C1-S67, C1-G66, C1-D65, C1-K64, C1-A63, C1-E62, C1-M61,C1-R60, C1-G59, C1-E58, C1-K57, C1-A56, C1-K55, C1-V54, C1-E53, C1-G52,C1-E51, C1-H50, C1-Q49, C1-R48, C1-R47, C1-F46, C1-E45, C1-S44, C1-E43,C1-D42, C1-Q41, C1-F40, C1-E39, C1-E38, C1-K37, C1-K36, C1-E35, C1-A34,C1-G33, C1-E32, C1-E31, C1-D30, C1-E29, C1-N28, C1-E27, C1-V26, C1-C25,C1-D24, C1-K23, C1-V22, C1-R21, C1-V20, C1-E19, C1-L18, C1-S17, C1-H16,C1-R15, C1-K14, C1-V13, C1-N12, C1-Y11, C1-L10, C1-L9, C1-A8, and/orC1-H7 of SEQ ID NO:31. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal HGPRBMY8 TM5-6 intertransmembrane domaindeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0198] In preferred embodiments, the following N-terminal HGPRBMY8 TM6-7intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: A1-Q15, V2-Q15, L3-Q15, A4-Q15, V5-Q15, W6-Q15,V7-Q15, D8-Q15, and/or V9-Q15 of SEQ ID NO:32. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these N-terminal HGPRBMY8 TM6-7intertransmembrane domain deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0199] In preferred embodiments, the following C-terminal HGPRBMY8 TM6-7intertransmembrane domain deletion polypeptides are encompassed by thepresent invention: A1-Q15, A1-P14, A1-V13, A1-Q12, A1-T11, A1-E10,A1-V9, A1-D8, and/or A1-V7 of SEQ ID NO:32. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these C-terminal HGPRBMY8 TM6-7intertransmembrane domain deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0200] The HGPRBMY8 polypeptide was predicted to comprise eight PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499 (1985); which are hereby incorporated by referenceherein.

[0201] In preferred embodiments, the following PKC phosphorylation sitepolypeptides are encompassed by the present invention: STCTNSTRESNSS(SEQ ID NO:76), QLLQVTNRFIFNL (SEQ ID NO:77), YPSKMTQRRGYLL (SEQ IDNO:78), EAKDGSLKAKEGS (SEQ ID NO:79), EGKEGSTKVEENS (SEQ ID NO:80),KVEENSMKADKGR (SEQ ID NO:81), ESLPPSRRNSNSN (SEQ ID NO:82), and/orGYMHKTIKKEIQD (SEQ ID NO:83). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of the HGPRBMY8 PKC phosphorylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0202] The HGPRBMY8 polypeptide was predicted to comprise five caseinkinase II phosphorylation sites using the Motif algorithm (GeneticsComputer Group, Inc.). Casein kinase II (CK-2) is a proteinserine/threonine kinase whose activity is independent of cyclicnucleotides and calcium. CK-2 phosphorylates many different proteins.The substrate specificity [1] of this enzyme can be summarized asfollows: (1) Under comparable conditions Ser is favored over Thr.; (2)An acidic residue (either Asp or Glu) must be present three residuesfrom the C-terminal of the phosphate acceptor site; (3) Additionalacidic residues in positions +1, +2, +4, and +5 increase thephosphorylation rate. Most physiological substrates have at least oneacidic residue in these positions; (4) Asp is preferred to Glu as theprovider of acidic determinants; and (5) A basic residue at theN-terminal of the acceptor site decreases the phosphorylation rate,while an acidic one will increase it.

[0203] A consensus pattern for casein kinase II phosphorylations site isas follows: [ST]-x(2)-[DE], wherein ‘x’ represents any amino acid, and Sor T is the phosphorylation site.

[0204] Additional information specific to aminoacyl-transfer RNAsynthetases class-II domains may be found in reference to the followingpublication: Pinna L. A., Biochim. Biophys. Acta 1054:267-284 (1990);which is hereby incorporated herein in its entirety.

[0205] In preferred embodiments, the following casein kinase IIphosphorylation site polypeptide is encompassed by the presentinvention: STCTNSTRESNSSH (SEQ ID NO:84), TGTSESSVEARGSE (SEQ ID NO:85),GKEGSTKVEENSMK (SEQ ID NO:86), DDINFSEDDVEAVN (SEQ ID NO:87), and/orPPKEDSHPDLPGTE (SEQ ID NO:88). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of this casein kinase II phosphorylation site polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0206] The HGPRBMY8 polypeptide was predicted to comprise two cAMP- andcGMP-dependent protein kinase phosphorylation site using the Motifalgorithm (Genetics Computer Group, Inc.). There has been a number ofstudies relative to the specificity of cAMP- and cGMP-dependent proteinkinases. Both types of kinases appear to share a preference for thephosphorylation of serine or threonine residues found close to at leasttwo consecutive N-terminal basic residues.

[0207] A consensus pattern for cAMP- and cGMP-dependent protein kinasephosphorylation sites is as follows: [RK](2)-x-[ST], wherein “x”represents any amino acid, and S or T is the phosphorylation site.

[0208] Additional information specific to cAMP- and cGMP-dependentprotein kinase phosphorylation sites may be found in reference to thefollowing publication: Fremisco J. R., Glass D. B., Krebs E. G, J. Biol.Chem. 255:4240-4245 (1980); Glass D. B., Smith S. B., J. Biol. Chem.258:14797-14803 (1983); and Glass D. B., El-Maghrabi M. R., Pilkis S.J., J. Biol. Chem. 261:2987-2993 (1986); which is hereby incorporatedherein in its entirety.

[0209] In preferred embodiments, the following cAMP- and cGMP-dependentprotein kinase phosphorylation site polypeptide is encompassed by thepresent invention: LLYNVKRHSLEVRV (SEQ ID NO:89), and/or SLPPSRRNSNSNPP(SEQ ID NO:90). Polynucleotides encoding this polypeptide are alsoprovided. The present invention also encompasses the use of this cAMP-and cGMP-dependent protein kinase phosphorylation site polypeptide as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0210] The HGPRBMY8 polypeptide has been shown to comprise threeglycosylation sites according to the Motif algorithm (Genetics ComputerGroup, Inc.). As discussed more specifically herein, proteinglycosylation is thought to serve a variety of functions including:augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

[0211] Asparagine glycosylation sites have the following consensuspattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site.However, it is well known that that potential N-glycosylation sites arespecific to the consensus sequence Asn-Xaa-Ser/Thr. However, thepresence of the consensus tripeptide is not sufficient to conclude thatan asparagine residue is glycosylated, due to the fact that the foldingof the protein plays an important role in the regulation ofN-glycosylation. It has been shown that the presence of proline betweenAsn and Ser/Thr will inhibit N-glycosylation; this has been confirmed bya recent statistical analysis of glycosylation sites, which also showsthat about 50% of the sites that have a proline C-terminal to Ser/Thrare not glycosylated. Additional information relating to asparagineglycosylation may be found in reference to the following publications,which are hereby incorporated by reference herein: Marshall R. D., Annu.Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl.Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336(1983); Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); andMiletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).

[0212] In preferred embodiments, the following asparagine glycosylationsite polypeptides are encompassed by the present invention:TSTCTNSTRESNSS (SEQ ID NO:91), STRESNSSHTCMPL (SEQ ID NO:92), and/orGEDDINFSEDDVEA (SEQ ID NO:93). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these HGPRBMY8 asparagine glycosylation site polypeptide asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0213] The HGPRBMY8 polypeptide was predicted to comprise eightN-myristoylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). An appreciable number of eukaryotic proteins are acylatedby the covalent addition of myristate (a C14-saturated fatty acid) totheir N-terminal residue via an amide linkage. The sequence specificityof the enzyme responsible for this modification, myristoyl CoA:proteinN-myristoyl transferase (NMT), has been derived from the sequence ofknown N-myristoylated proteins and from studies using syntheticpeptides. The specificity seems to be the following: i.) The N-terminalresidue must be glycine; ii.) In position 2, uncharged residues areallowed; iii.) Charged residues, proline and large hydrophobic residuesare not allowed; iv.) In positions 3 and 4, most, if not all, residuesare allowed; v.) In position 5, small uncharged residues are allowed(Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) Inposition 6, proline is not allowed.

[0214] A consensus pattern for N-myristoylation is as follows:G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein ‘x’ represents any amino acid,and G is the N-myristoylation site.

[0215] Additional information specific to N-myristoylation sites may befound in reference to the following publication: Towler D. A., Gordon J.I., Adams S. P., Glaser L., Annu. Rev. Biochem. 57:69-99 (1988); andGrand R. J. A., Biochem. J. 258:625-638 (1989); which is herebyincorporated herein in its entirety.

[0216] In preferred embodiments, the following N-myristoylation sitepolypeptides are encompassed by the present invention: ISLAHGIIRSTVLVIF(SEQ ID NO:94), CSMIWGASPSYTILSV (SEQ ID NO:95), MEAKDGSLKAKEGSTG (SEQID NO:96), LKAKEGSTGTSESSVE (SEQ ID NO:97), KEGSTGTSESSVEARG (SEQ IDNO:98), TVASDGSMEGKEGSTK (SEQ ID NO:99), HPDLPGTEGGTEGKIV (SEQ IDNO:100), and/or LPGTEGGTEGKIVPSY (SEQ ID NO:101). The present inventionalso encompasses the use of these N-myristoylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0217] Moreover, in confirmation of HGPRBMY8 representing a novel GPCR,the HGPRBMY8 polypeptide was predicted to comprise a G-protein coupledreceptor motif using the Motif algorithm (Genetics Computer Group,Inc.). G-protein coupled receptors (also called R7G) are an extensivegroup of hormones, neurotransmitters, odorants and light receptors whichtransduce extracellular signals by interaction with guaninenucleotide-binding (G) proteins. Some examples of receptors that belongto this family are provided as follows: 5-hydroxytryptamine (serotonin)1A to 1F, 2A to 2C, 4, 5A, 5B, 6 and 7, Acetylcholine, muscarinic-type,M1 to M5, Adenosine A1, A2A, A2B and A3, Adrenergic alpha-1A to -1C;alpha-2A to -2D; beta-1 to -3, Angiotensin II types I and II, Bombesinsubtypes 3 and 4, Bradykinin B1 and B2, c3a and C5a anaphylatoxin,Cannabinoid CB1 and CB2, Chemokines C-C CC-CKR-1 to CC-CKR-8, ChemokinesC-X-C CXC-CKR-1 to CXC-CKR-4, Cholecystokinin-A andcholecystokinin-B/gastrin, Dopamine D1 to D5, Endothelin ET-a and ET-b,fMet-Leu-Phe (fMLP) (N-formyl peptide), Follicle stimulating hormone(FSH-R), Galanin, Gastrin-releasing peptide (GRP-R),Gonadotropin-releasing hormone (GNRH-R), Histamine H1 and H2 (gastricreceptor I), Lutropin-choriogonadotropic hormone (LSH-R), MelanocortinMC1R to MC5R, Melatonin, Neuromedin B (NMB-R), Neuromedin K (NK-3R),Neuropeptide Y types 1 to 6, Neurotensin (NT-R), Octopamine (tyramine)from insects, Odorants, Opioids delta-, kappa- and mu-types, Oxytocin(OT-R), Platelet activating factor (PAF-R), Prostacyclin, ProstaglandinD2, Prostaglandin E2, EP1 to EP4 subtypes, Prostaglandin F2,Purinoreceptors (ATP), Somatostatin types 1 to 5, Substance-K (NK-2R),Substance-P (NK-1R), Thrombin, Thromboxane A2, Thyrotropin (TSH-R),Thyrotropin releasing factor (TRH-R), Vasopressin V1a, V1b and V2,Visual pigments (opsins and rhodopsin), Proto-oncogene mas,Caenorhabditis elegans putative receptors C06G4.5, C38C10.1, C43C3.2,T27D1.3 and ZC84.4, Three putative receptors encoded in the genome ofcytomegalovirus: US27, US28, and UL33., ECRF3, a putative receptorencoded in the genome of herpesvirus saimiri.

[0218] The structure of all GPCRs are thought to be identical. They haveseven hydrophobic regions, each of which most probably spans themembrane. The N-terminus is located on the extracellular side of themembrane and is often glycosylated, while the C-terminus is cytoplasmicand generally phosphorylated. Three extracellular loops alternate withthree intracellular loops to link the seven transmembrane regions. Most,but not all of these receptors, lack a signal peptide. The mostconserved parts of these proteins are the transmembrane regions and thefirst two cytoplasmic loops. A conserved acidic-Arg-aromatic triplet ispresent in the N-terminal extremity of the second cytoplasmic loop andcould be implicated in the interaction with G proteins.

[0219] The putative consensus sequence for GPCRs comprises the conservedtriplet and also spans the major part of the third transmembrane helix,and is as follows:[GSTALIVMFYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-[LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R-[FYWCSH]-x(2)-[LIVM],where “X” represents any amino acid.

[0220] Additional information relating to G-protein coupled receptorsmay be found in reference to the following publications: Strosberg A.D., Eur. J. Biochem. 196:1-10 (1991); Kerlavage A. R., Curr. Opin.Struct. Biol. 1:394-401 (1991); Probst W. C., Snyder L. A., Schuster D.I., Brosius J., Sealfon S. C., DNA Cell Biol. 11:1-20 (1992); SavareseT. M., Fraser C. M., Biochem. J. 283:1-9 (1992); Branchek T., Curr.Biol. 3:315-317 (1993); Stiles G. L., J. Biol. Chem. 267:6451-6454(1992); Friell T., Kobilka B. K., Lefkowitz R. J., Caron M. G., TrendsNeurosci. 11:321-324 (1988); Stevens C. F., Curr. Biol. 1:20-22 (1991);Sakurai T., Yanagisawa M., Masaki T., Trends Pharmacol. Sci. 13:103-107(1992); Salesse R., Remy J. J., Levin J. M., Jallal B., Gamier J.,Biochimie 73:109-120 (1991); Lancet D., Ben-Arie N., Curr. Biol.3:668-674 (1993); Uhl G. R., Childers S., Pasternak G., Trends Neurosci.17:89-93 (1994); Barnard E. A., Burnstock G., Webb T. E., TrendsPharmacol. Sci. 15:67-70 (1994); Applebury M. L., Hargrave P. A., VisionRes. 26:1881-1895 (1986); Attwood T. K., Eliopoulos E. E., Findlay J. B.C., Gene 98:153-159 (1991); http://www.gcrdb.uthscsa.edu/; andhttp://swift.embl-heidelberg.de/7tm/.

[0221] In preferred embodiments, the following G-protein coupledreceptors signature polypeptide is encompassed by the present invention:SVVSFIVIPLIVMIACYSVVF (SEQ ID NO: 102). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of the HGPRBMY8 G-protein coupled receptors signaturepolypeptide as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0222] For the production of antibodies, various hosts including goats,rabbits, sheep, rats, mice, humans, and others, can be immunized byinjection with HGPRBMY8 polypeptide, or any fragment or oligopeptidethereof, which has immunogenic properties. Depending on the hostspecies, various adjuvants may be used to increase the immunologicalresponse. Non-limiting examples of suitable adjuvants include Freund's(complete and incomplete), mineral gels such as aluminum hydroxide orsilica, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Adjuvants typically used in humans include BCG (bacilli Calmette Guérin)and Corynebacterium parvumn.

[0223] Preferably, the peptides, fragments, or oligopeptides used toinduce antibodies to HGPRBMY8 polypeptide (i.e., immunogens) have anamino acid sequence having at least five amino acids, and morepreferably, at least 7-10 amino acids. It is also preferable that theimmunogens are identical to a portion of the amino acid sequence of thenatural protein; they may also contain the entire amino acid sequence ofa small, naturally occurring molecule. The peptides, fragments oroligopeptides may comprise a single epitope or antigenic determinant ormultiple epitopes. Short stretches of HGPRBMY8 amino acids may be fusedwith those of another protein, such as KLH, and antibodies are producedagainst the chimeric molecule.

[0224] Monoclonal antibodies to HGPRBMY8 polypeptide, or immunogenicfragments thereof, may be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma technique,the human B-cell hybridoma technique, and the EBV-hybridoma technique(G. Kohler et al., 1975, Nature, 256:495-497; D. Kozbor et al., 1985, J.Immunol. Methods, 81:31-42; R. J. Cote et al., 1983, Proc. Natl. Acad.Sci. USA, 80:2026-2030; and S. P. Cole et al., 1984, Mol. Cell Biol.,62:109-120). The production of monoclonal antibodies is well known androutinely used in the art.

[0225] In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (S. L. Morrison et al., 1984, Proc.Natl. Acad. Sci. USA, 81:6851-6855; M. S. Neuberger et al., 1984,Nature, 312:604-608; and S. Takeda et al., 1985, Nature, 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceHGPRBMY8 polypeptide-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries (D. R. Burton, 1991, Proc. Natl. Acad. Sci. USA, 88:11120-3).Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (R. Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA,86:3833-3837 and G. Winter et al., 1991, Nature, 349:293-299).

[0226] Antibody fragments, which contain specific binding sites forHGPRBMY8 polypeptide, may also be generated. For example, such fragmentsinclude, but are not limited to, F(ab′)₂ fragments which can be producedby pepsin digestion of the antibody molecule and Fab fragments which canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (W. D. Huse et al., 1989, Science, 254.1275-1281).

[0227] Various immunoassays can be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve measuring the formationof complexes between HGPRBMY8 polypeptide and its specific antibody. Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive with two non-interfering HGPRBMY8 polypeptide epitopes ispreferred, but a competitive binding assay may also be employed (Maddox,supra).

[0228] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with HGPRBMY8 polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2. Yetanother aspect of the invention relates to a method of inducingimmunological response in a mammal which comprises, delivering HGPRBMY8polypeptide via a vector directing expression of HGPRBMY8 polynucleotidein vivo in order to induce such an immunological response to produceantibody to protect said animal from diseases.

[0229] A further aspect of the invention relates to animmunological/vaccine formulation (composition) which, when introducedinto a mammalian host, induces an immunological response in that mammalto an HGPRBMY8 polypeptide wherein the composition comprises an HGPRBMY8polypeptide or HGPRBMY8 gene. The vaccine formulation may furthercomprise a suitable carrier. Since the HGPRBMY8 polypeptide may bebroken down in the stomach, it is preferably administered parenterally(including subcutaneous, intramuscular, intravenous, intradermal, etc.,injection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials, and maybe stored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in-water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

[0230] In an embodiment of the present invention, the polynucleotideencoding the HGPRBMY8 polypeptide, or any fragment or complementthereof, may be used for therapeutic purposes. In one aspect, antisense,to the polynucleotide encoding the HGPRBMY8 polypeptide, may be used insituations in which it would be desirable to block the transcription ofthe mRNA. In particular, cells may be transformed with sequencescomplementary to polynucleotides encoding HGPRBMY8 polypeptide. Thus,complementary molecules may be used to modulate HGPRBMY8 polynucleotideand polypeptide activity, or to achieve regulation of gene function.Such technology is now well known in the art, and sense or antisenseoligomers or oligonucleotides, or larger fragments, can be designed fromvarious locations along the coding or control regions of polynucleotidesequences encoding HGPRBMY8 polypeptide.

[0231] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods, which are well known to those skilled in the art,can be used to construct recombinant vectors which will express anucleic acid sequence that is complementary to the nucleic acid sequenceencoding the HGPRBMY8 polypeptide. These techniques are described bothin J. Sambrook et al., supra and in F. M. Ausubel et al., supra.

[0232] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy”. Thus for example, cells from a subject may be engineered witha polynucleotide, such as DNA or RNA, to encode a polypeptide ex vivo,and for example, by the use of a retroviral plasmid vector. The cellscan then be introduced into the subject.

[0233] The genes encoding the HGPRBMY8 polypeptide can be turned off bytransforming a cell or tissue with an expression vector that expresseshigh levels of an HGPRBMY8 polypeptide-encoding polynucleotide, or afragment thereof. Such constructs may be used to introduceuntranslatable sense or antisense sequences into a cell. Even in theabsence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and even longer if appropriate replicationelements are designed to be part of the vector system.

[0234] Modifications of gene expression can be obtained by designingantisense molecules or complementary nucleic acid sequences (DNA, RNA,or PNA), to the control, 5′, or regulatory regions of the gene encodingthe HGPRBMY8 polypeptide, (e.g., signal sequence, promoters, enhancers,and introns). Oligonucleotides derived from the transcription initiationsite, e.g., between positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using “triple helix”base-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described (see, for example, J. E. Gee et al., 1994, In: B. E.Huber and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecule orcomplementary sequence may also be designed to block translation of mRNAby preventing the transcript from binding to ribosomes.

[0235] Ribozymes, i.e., enzymatic RNA molecules, may also be used tocatalyze the specific cleavage of RNA. The mechanism of ribozyme actioninvolves sequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Suitableexamples include engineered hammerhead motif ribozyme molecules that canspecifically and efficiently catalyze endonucleolytic cleavage ofsequences encoding HGPRBMY8 polypeptide.

[0236] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0237] Complementary ribonucleic acid molecules and ribozymes accordingto the invention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. Such methods include techniques forchemically synthesizing oligonucleotides, for example, solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding HGPRBMY8. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP. Alternatively, the cDNA constructs that constitutively or induciblysynthesize complementary RNA can be introduced into cell lines, cells,or tissues.

[0238] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl, rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytosine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0239] Many methods for introducing vectors into cells or tissues areavailable and are equally suitable for use in vivo, in vitro, and exvivo. For ex vivo therapy, vectors may be introduced into stem cellstaken from the patient and clonally propagated for autologous transplantback into that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods, which are well known in theart.

[0240] Any of the therapeutic methods described above may be applied toany individual in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0241] A further embodiment of the present invention embraces theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, diluent, or excipient, for any ofthe above-described therapeutic uses and effects. Such pharmaceuticalcompositions may comprise HGPRBMY8 nucleic acid, polypeptide, orpeptides, antibodies to HGPRBMY8 polypeptide, mimetics, agonists,antagonists, or inhibitors of HGPRBMY8 polypeptide or polynucleotide.The compositions may be administered alone, or in combination with atleast one other agent, such as a stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, hormones, or biological responsemodifiers.

[0242] The pharmaceutical compositions for use in the present inventioncan be administered by any number of routes including, but not limitedto, oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, orrectal means.

[0243] In addition to the active ingredients (i.e., the HGPRBMY8 nucleicacid or polypeptide, or functional fragments thereof), thepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers or excipients comprising auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Further details on techniques forformulation and administration are provided in the latest edition ofRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).

[0244] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0245] Pharmaceutical preparations for oral use can be obtained by thecombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropyl-methylcellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth, andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a physiologically acceptable saltthereof, such as sodium alginate.

[0246] Dragee cores may be used in conjunction with physiologicallysuitable coatings, such as concentrated sugar solutions, which may alsocontain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for product identification,or to characterize the quantity of active compound, i.e., dosage.

[0247] Pharmaceutical preparations, which can be used orally, includepush-fit capsules made of gelatin, as well as soft, scaled capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0248] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances, which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. In addition, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyloleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0249] For topical or nasal administration, penetrants or permeationagents that are appropriate to the particular barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

[0250] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0251] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Saltstend to be more soluble in aqueous solvents, or other protonic solvents,than are the corresponding free base forms. In other cases, thepreferred preparation may be a lyophilized powder which may contain anyor all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7%mannitol, at a pH range of 4.5 to 5.5, combined with a buffer prior touse. After the pharmaceutical compositions have been prepared, they canbe placed in an appropriate container and labeled for treatment of anindicated condition. For administration of HGPRBMY8 product, suchlabeling would include amount, frequency, and method of administration.

[0252] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose. Thedetermination of an effective dose or amount is well within thecapability of those skilled in the art. For any compound, thetherapeutically effective dose can be estimated initially either in cellculture assays, e.g., using neoplastic cells, or in animal models,usually mice, rabbits, dogs, or pigs. The animal model may also be usedto determine the appropriate concentration range and route ofadministration. Such information can then be used and extrapolated todetermine useful doses and routes for administration in humans.

[0253] A therapeutically effective dose refers to that amount of activeingredient, for example, HGPRBMY8 polypeptide, or fragments thereof,antibodies to HGPRBMY8 polypeptide, agonists, antagonists or inhibitorsof HGPRBMY8 polypeptide, which ameliorates, reduces, or eliminates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, which can be expressed as the ratio, LD₅₀/ED₅₀.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in determining a range of dosages for human use. Preferreddosage contained in a pharmaceutical composition is within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0254] The practitioner, who will consider the factors related to theindividual requiring treatment, will determine the exact dosage. Dosageand administration are adjusted to provide sufficient levels of theactive moiety or to maintain the desired effect. Factors, which may betaken into account, include the severity of the individual's diseasestate, general health of the patient, age, weight, and gender of thepatient, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. As a general guide, long-acting pharmaceutical compositions maybe administered every 3 to 4 days, every week, or once every two weeks,depending on half-life and clearance rate of the particular formulation.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

[0255] Normal dosage amounts may vary from 0.1 to 100,000 micrograms(μg), up to a total dose of about 1 gram (g), depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and is generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, and the like.

[0256] In another embodiment of the present invention, antibodies whichspecifically bind to the HGPRBMY8 polypeptide may be used for thediagnosis of conditions or diseases characterized by expression (oroverexpression) of the HGPRBMY8 polynucleotide or polypeptide, or inassays to monitor patients being treated with the HGPRBMY8 polypeptide,or its agonists, antagonists, or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for use in therapeutic methods. Diagnostic assays forthe HGPRBMY8 polypeptide include methods, which utilize the antibody anda label to detect the protein in human body fluids or extracts of cellsor tissues. The antibodies may be used with or without modification, andmay be labeled by joining them, either covalently or non-covalently,with a reporter molecule. A wide variety of reporter molecules, whichare known in the art, may be used, several of which are described above.In particular, a method of detecting a G-protein coupled receptor,homologue, or an antibody-reactive fragment thereof, in a sample,comprising: a) contacting the sample with an antibody specific for thepolypeptide, or an antigenic fragment thereof, under conditions in whichan antigen-antibody complex can form between the antibody and thepolypeptide or antigenic fragment thereof in the sample; and b)detecting an antigen-antibody complex formed in step (a), whereindetection of the complex indicates the presence of an antigenic fragmentthereof, in the sample.

[0257] The use of mammalian cell reporter assays to demonstratefunctional coupling of known GPCRs (G Protein Coupled Receptors) hasbeen well documented in the literature (Gilman, 1987, Boss et al., 1996;Alam & Cook, 1990; George et al., 1997; Selbie & Hill, 1998; Rees etal., 1999). In fact, reporter assays have been successfully used foridentifying novel small molecule agonists or antagonists against GPCRsas a class of drug targets (Zlokarnik et al., 1998; George et al., 1997;Boss et al., 1996; Rees et al, 2001). In such reporter assays, apromoter is regulated as a direct consequence of activation of specificsignal transduction cascades following agonist binding to a GPCR (Alam &Cook 1990; Selbie & Hill, 1998; Boss et al., 1996; George et al., 1997;Gilman, 1987).

[0258] A number of response element-based reporter systems have beendeveloped that enable the study of GPCR function. These include cAMPresponse element (CRE)-based reporter genes for G alpha i/o, G alphas-coupled GPCRs, Nuclear Factor Activator of Transcription (NFAT)-basedreporters for G alpha q/11 11 or the promiscuous G protein G alpha 15/16-coupled receptors and MAP kinase reporter genes for use in Galpha i/ocoupled receptors (Selbie & Hill, 1998; Boss et al., 1996; George etal., 1997; Blahos, et al., 2001; Offermann & Simon, 1995; Gilman, 1987;Rees et al., 2001). Transcriptional response elements that regulate theexpression of Beta-Lactamase within a CHO K1 cell line (CHO-NFAT/CRE:Aurora Biosciences™) (Zlokarnik et al., 1998) have been implemented tocharacterize the function of the orphan HGPRBMY8 polypeptide of thepresent invention. The system enables demonstration of constitutiveG-protein coupling to endogenous cellular signaling components uponintracellular overexpression of orphan receptors. Overexpression hasbeen shown to represent a physiologically relevant event. For example,it has been shown that overexpression occurs in nature during metastaticcarcinomas, wherein defective expression of the monocyte chemotacticprotein 1 receptor, CCF2, in macrophages is associated with theincidence of human ovarian carcinoma (Sica, et al.,2000; Salcedo et al.,2000). Indeed, it has been shown that overproduction of the Beta 2Adrenergic Receptor in transgenic mice leads to constitutive activationof the receptor signaling pathway such that these mice exhibit increasedcardiac output (Kypson et al., 1999; Dorn et al., 1999). These are onlya few of the many examples demonstrating constitutive activation ofGPCRs whereby many of these receptors are likely to be in the active,R*, conformation (J. Wess 1997) (see Example 7).

[0259] Several assay protocols including ELISA, RIA, and FACS formeasuring HGPRBMY8 polypeptide are known in the art and provide a basisfor diagnosing altered or abnormal levels of HGPRBMY8 polypeptideexpression. Normal or standard values for HGPRBMY8 polypeptideexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody tothe HGPRBMY8 polypeptide under conditions suitable for complexformation. The amount of standard complex formation may be quantified byvarious methods; photometric means are preferred. Quantities of HGPRBMY8polypeptide expressed in subject sample, control sample, and diseasesamples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease.

Microarrays and Screening Assays

[0260] In another embodiment of the present invention, oligonucleotides,or longer fragments derived from the HGPRBMY8 polynucleotide sequencedescribed herein may be used as targets in a microarray. The microarraycan be used to monitor the expression level of large numbers of genessimultaneously (to produce a transcript image), and to identify geneticvariants, mutations and polymorphisms. This information may be used todetermine gene function, to understand the genetic basis of a disease,to diagnose disease, and to develop and monitor the activities oftherapeutic agents. In a particular aspect, the microarray is preparedand used according to the methods described in WO 95/11995 (Chee etal.); D. J. Lockhart et al., 1996, Nature Biotechnology, 14:1675-1680;and M. Schena et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-10619).Microarrays are further described in U.S. Pat. No. 6,015,702 to P. Lalet al.

[0261] In another embodiment of this invention, the nucleic acidsequence, which encodes the HGPRBMY8 polypeptide, may also be used togenerate hybridization probes, which are useful for mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions (HACs), yeast artificial chromosomes(YACs), bacterial artificial chromosomes (BACs), bacterial PIconstructions, or single chromosome cDNA libraries, as reviewed by C. M.Price, 1993, Blood Rev., 7:127-134 and by B. J. Trask, 1991, TrendsGenet., 7:149-154.

[0262] Fluorescent In Situ Hybridization (FISH), (as described in I.Verma et al., 1988, Human Chromosomes: A Manual of Basic TechniquesPergamon Press, New York, N.Y.) may be correlated with other physicalchromosome mapping techniques and genetic map data. Examples of geneticmap data can be found in numerous scientific journals, or at OnlineMendelian Inheritance in Man (OMIM). Correlation between the location ofthe gene encoding the HGPRBMY8 polypeptide on a physical chromosomal mapand a specific disease, or predisposition to a specific disease, mayhelp delimit the region of DNA associated with that genetic disease. Thenucleotide sequences, particularly that of SEQ ID NO:1, or fragmentsthereof, according to this invention may be used to detect differencesin gene sequences between normal, carrier, or affected individuals.

[0263] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers, even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (R. A. Gatti etal., 1988, Nature, 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the present invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,and the like, among normal, carrier, or affected individuals.

[0264] In another embodiment of the present invention, the HGPRBMY8polypeptide, its catalytic or immunogenic fragments or oligopeptidesthereof, can be used for screening libraries of compounds in any of avariety of drug screening techniques. The fragment employed in suchscreening may be free in solution, affixed to a solid support, borne ona cell surface, or located intracellularly. The formation of bindingcomplexes, between HGPRBMY8 polypeptide, or portion thereof, and theagent being tested, may be measured utilizing techniques commonlypracticed in the art. In particular, a method of screening a library ofmolecules or compounds with an HGPRBMY8 polynucleotide, or fragmentthereof, to identify at least one molecule or compound therein whichspecifically binds to the G-protein coupled receptor polynucleotidesequence, preferably the HGPRBMY8 polynucleotide sequence, or fragmentthereof, comprising: a) combining the G-protein coupled receptorpolynucleotide, or fragment thereof, with a library of molecules orcompounds under conditions to allow specific binding; and b) detectingspecific binding, thereby identifying a molecule or compound, whichspecifically binds to a G-protein coupled receptor-encodingpolynucleotide sequence. In a further embodiment, the screening methodis a high throughput screening method. Preferably, the library isselected from the group consisting of DNA molecules, RNA molecules,artificial chromosome constructions, PNAs, peptides and proteins. Inanother preferred embodiment, the candidate small molecules or compoundsare a drug or therapeutic.

[0265] In yet another embodiment, a method of screening for candidatecompounds capable of modulating activity of a G-protein coupledreceptor-encoding polypeptide, comprising: a) contacting a test compoundwith a cell or tissue expressing the G-protein coupled receptorpolypeptide, homologue, or fragment thereof; and b) selecting ascandidate modulating compounds those test compounds that modulateactivity of the G-protein coupled receptor polypeptide. Preferably, thecandidate compounds are agonists or antagonists of G-protein coupledreceptor activity. More preferably, the polypeptide activity isassociated with the brain.

[0266] Another technique for drug screening, which may be used, providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in WO 84/03564 (Venton,et al.). In this method, as applied to the HGPRBMY8 protein, largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with the HGPRBMY8 polypeptide, or fragmentsthereof, and washed. Bound HGPRBMY8 polypeptide is then detected bymethods well known in the art. Purified HGPRBMY8 polypeptide can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0267] In a further embodiment of this invention, competitive drugscreening assays can be used in which neutralizing antibodies, capableof binding the HGPRBMY8 polypeptide, specifically compete with a testcompound for binding to the HGPRBMY8 polypeptide. In this manner, theantibodies can be used to detect the presence of any peptide, whichshares one or more antigenic determinants with the HGPRBMY8 polypeptide.

[0268] Other screening and small molecule (e.g., drug) detection assayswhich involve the detection or identification of small molecules orcompounds that can bind to a given protein, i.e., the HGPRBMY8polypeptide, are encompassed by the present invention. Particularlypreferred are assays suitable for high throughput screeningmethodologies. In such binding-based screening or detection assays, afunctional assay is not typically required. All that is needed is atarget protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) to bescreened or assayed for binding to the protein target. Preferably, mostsmall molecules that bind to the target protein will modulate activityin some manner, due to preferential, higher affinity binding tofunctional areas or sites on the protein.

[0269] An example of such an assay is the fluorescence based thermalshift assay (3-Dimensional Pharmaceuticals, Inc., 3DP; Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, HGPRBMY8 polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

EXAMPLES

[0270] The Examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the scope ofthe invention in any way. The Examples do not include detaileddescriptions for conventional methods employed, such as in theconstruction of vectors, the insertion of cDNA into such vectors, or theintroduction of the resulting vectors into the appropriate host. Suchmethods are well known to those skilled in the art and are described innumerous publications, for example, Sambrook, Fritsch, and Maniatis,Molecular Cloning: a Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Laboratory Press, USA, (1989).

Example 1 Bioinformatics Analysis

[0271] G-protein coupled receptor sequences were used as a probes tosearch human genomic sequence databases. The search program used wasgapped BLAST (S. F. Altschul, et al., Nuc. Acids Res., 25:3389-4302(1997)). The top genomic exon hits from the BLAST results were searchedback against the non-redundant protein and patent sequence databases.From this analysis, exons encoding potential full-length sequence of anovel human GPCR, HGPRBMY8, was identified directly from the genomicsequence. The full-length clone of this GPCR was experimentally obtainedby RT-PCR using the sequence from genomic data. The complete proteinsequence of HGPRBMY8 was analyzed for potential transmembrane domains.TMPRED program (K. Hofmann and W. Stoffel, Biol. Chem., 347:166 (1993)was used for transmembrane prediction. The program predicted seventransmembrane domains and the predicted domains match with the predictedtransmembrane domains of related GPCRs at the sequence level. Based onsequence, structure and known GPCR signature sequences, the orphanprotein, HGPRBMY8 of the present invention, is a novel human GPCR.

Example 2 Cloning of the Novel Human GPCR HGPRBMY8

[0272] HGPRBMY8 was cloned from a human brain cDNA library (Clontech;Palo Alto, Calif.) by PCR amplification of the predicted cDNA sequenceusing sequence specific oligonucleotides. The 5′ sense oligonucleotidewas as follows:

[0273] 5′-GGCCGAATTCGCAACCTGTCTCACGCCCTCTGG-3′ (SEQ ID NO:5). The 3′anti-sense oligonucleotide was as follows:

[0274] 5′-GGCCGAATTCGGACAGTTCAAGGTTTGCCTTAGAAC-3′ (SEQ ID NO:6). Theseoligonucleotides contained EcoRI restriction enzyme sites for subcloningthe PCR fragment into the mammalian expression vector, pcDNA6. Samplescontaining human brain cDNA, the 5 prime sense, and 3 prime anti-senseoligonucleotides were subjected to PCR amplification followed by gelpurification of the amplified product. The inserts of cDNA clones thatwere positive by PCR were sized, and two of the largest clones (˜1.6 Kb)were sequenced using conventional sequencing methods. Purified samplewas digested with EcoRI, extracted with phenol:chloroform, and ligatedinto pcDNA6. The resultant plasmids were subjected to DNA sequencing andthe sequences were verified by comparison with the database sample.

Example 3 Expression Profiling of Novel Human GPCR, HGPRBMY8

[0275] The oligonucleotides used for the expression profiling ofHGPRBMY8 are:

[0276] HGPRBMY8-2s: 5′-GCAGAGCACTCCTCCACTCT-3′ (SEQ ID NO:34)

[0277] HGPRBMY8-2a: 5′-AGCAGGCAATCATGACAATC-3′ (SEQ ID NO:35)

[0278] These oligonucleotides were used to measure the steady statelevels of mRNA by quantitative PCR. Briefly, first strand cDNA was madefrom commercially available mRNA (Clontech; Palo Alto, Calif.). Therelative amount of cDNA used in each assay (2.5 ng of cDNA per assay)was determined by performing a parallel experiment using a primer pairfor the cyclophilin gene, which is expressed in equal amounts in alltissues. The cyclophilin primer pair detected small variations in theamount of cDNA in each sample, and these data were used fornormalization of the data obtained with the primer pair for HGPRBMY8.The PCR data were converted into a relative assessment of the differencein transcript abundance among the tissues tested and the data arepresented in FIG. 7. Transcripts corresponding to the orphan GPCR,HGPRBMY8, were found to be highly expressed in brain.

Example 4 G-protein Coupled Receptor PCR Expression Profiling

[0279] Based on HGPRBMY8's expression in the brain, further analysis wascarried out to determine if there was any additional specificity withinsub regions. The same PCR primer pair that was used to identify HGPRBMY8(also referred to as GPCR 58 and GPCR84) cDNA clones was used to measurethe steady state levels of mRNA by quantitative PCR.

[0280] GPCR84-s GTTAGCCTCACCCACCTGTT (SEQ ID NO:36)

[0281] GPCR84-a CACAATCCAGGTGCCATAGA (SEQ ID NO:37)

[0282] Briefly, first strand cDNA was made from commercially availablebrain subregion mRNA (Clontech) and subjected to real time quantitativePCR using a PE 5700 instrument (Applied Biosystems; Foster City, Calif.)which detects the amount of DNA amplified during each cycle by thefluorescent output of SYBR green, a DNA binding dye specific for doublestrands. The specificity of the primer pair for its target is verifiedby performing a thermal denaturation profile at the end of the run whichgives an indication of the number of different DNA sequences present bydetermining melting Tm. In the case of the HGPRBMY8 primer pair, onlyone DNA fragment was detected having a homogeneous melting point.Contributions of contaminating genomic DNA to the assessment of tissueabundance is controlled for by performing the PCR with first strand madewith and without reverse transcriptase. In all cases, the contributionof material amplified in the no reverse transcriptase controls wasnegligible.

[0283] More specifically, since HGPRBMY8 is expressed at extremely lowlevels, each PCR reaction contained the amount of first strand cDNA madefrom 100 nanograms of poly A+ RNA (2.5 nanograms is the standardamount).

[0284] The number of reactions and amount of mix needed was firstdetermined. All of the samples were run in triplicate, so sample tubesneeded 3.5 reactions worth of mixture using the following formula as aguide (2× # tissue samples+1 no template control+1 for pipetting error)(3.5).

[0285] The reaction mixture consisted of the following components andvolumes: COMPONENTS VOL/RXN 2X SybrGreen Master Mix 25 microliters water23.5 microliters primer mix (10 μM ea.)  0.5 microliters cDNA (100ng/uL)  1 microliter

[0286] The mixture was initially made without cDNA for enough reactionsas determined above. The mix (171.5 μl) was then aliquoted into sampletubes. cDNA (3.5 μl) was added to each sample tube, mixed gently, andspun down for collection. Three 50 μl samples were aliquoted to theoptical plate, where the primer and sample were set up for sampleanalysis. The threshold was set in Log view to intersect linear regionsof amplification. The background was set in Linear view to 2-3 cyclesbefore the amplification curve appears. The mean values for RT+ wascalculated and normalized to Cyclophilin: dCt=sample mean−cyclophilinmean. The ddCt was determined by subtracting individual dCts from thehighest value of dCt in the list. The relative abundance was determinedby formula 2^ ddCt.

[0287] Small variations in the amount of cDNA used in each tube wasdetermined by performing a parallel experiment using a primer pair for agene expressed in equal amounts in all tissues, cyclophilin. These datawere used to normalize the data obtained with the HGPRBMY8 primer pair.The PCR data was converted into a relative assessment of the differencein transcript abundance amongst the tissues tested and the data arepresented in bar graph form. Transcripts corresponding to HGPRBMY8 areexpressed approximately 825 times greater in the caudate nucleus than inthe substantia nigra. Low level expression was detected in the thalamus,amygdala, hippocampus, cerebellum and corpus collosum (see FIG. 8).

Example 5 Signal Transduction Assays

[0288] The activity of GPCRs or homologues thereof, can be measuredusing any assay suitable for the measurement of the activity of a Gprotein-coupled receptor, as commonly known in the art. Signaltransduction activity of a G protein-coupled receptor can be monitor bymonitoring intracellular Ca²⁺, cAMP, inositol 1,4,5-triphosphate (IP₃),or 1,2-diacylglycerol (DAG). Assays for the measurement of intracellularCa²⁺ are described in Sakurai et al. (EP 480 381). Intracellular IP₃ canbe measured using a kit available from Amersham, Inc. (ArlingtonHeights, Ill.). A kit for measuring intracellular cAMP is available fromDiagnostic Products, Inc. (Los Angeles, Calif.).

[0289] Activation of a G protein-coupled receptor triggers the releaseof Ca²⁺ ions sequestered in the mitochondria, endoplasmic reticulum, andother cytoplasmic vesicles into the cytoplasm. Fluorescent dyes, e.g.,fura-2, can be used to measure the concentration of free cytoplasmicCa²⁺. The ester of fura-2, which is lipophilic and can diffuse acrossthe cell membrane, is added to the media of the host cells expressingGPCRs. Once inside the cell, the fura-2 ester is hydrolyzed by cytosolicesterases to its non-lipophilic form, and then the dye cannot diffuseback out of the cell. The non-lipophilic form of fura-2 will fluorescewhen it binds to free Ca²⁺. The fluorescence can be measured withoutlysing the cells at an excitation spectrum of 340 nm or 380 nm and atfluorescence spectrum of 500 nm (Sakurai et al., EP 480 381).

[0290] Upon activation of a G protein-coupled receptor, the rise of freecytosolic Ca²⁺ concentrations is preceded by the hydrolysis ofphosphatidylinositol 4,5-bisphosphate. Hydrolysis of this phospholipidby the phospholipase C yields 1,2-diacylglycerol (DAG), which remains inthe membrane, and water-soluble inositol 1,4,5-triphosphate (IP₃).Binding of ligands or agonists will increase the concentration of DAGand IP₃. Thus, signal transduction activity can be measured bymonitoring the concentration of these hydrolysis products.

[0291] To measure the IP₃ concentrations, radioactivity labeled³H-inositol is added to the media of host cells expressing GPCRs. The³H-inositol is taken up by the cells and incorporated into IP₃. Theresulting inositol triphosphate is separated from the mono anddi-phosphate forms and measured (Sakurai et al., EP 480 381).Alternatively, Amersham provides an inositol 1,4,5-triphosphate assaysystem. With this system Amersham provides tritylated inositol1,4,5-triphosphate and a receptor capable of distinguishing theradioactive inositol from other inositol phosphates. With these reagentsan effective and accurate competition assay can be performed todetermine the inositol triphosphate levels.

[0292] Cyclic AMP levels can be measured according to the methodsdescribed in Gilman et al., Proc. Natl. Acad. Sci. 67:305-312 (1970). Inaddition, a kit for assaying levels of cAMP is available from DiagnosticProducts Corp. (Los Angeles, Calif.).

Example 6 GPCR Activity

[0293] Another method for screening compounds which are antagonists, andthus inhibit activation of the receptor polypeptide of the presentinvention is provided. This involves determining inhibition of bindingof labeled ligand, such as dATP, dAMP, or UTP, to cells which have thereceptor on the surface thereof, or cell membranes containing thereceptor. Such a method further involves transfecting a eukaryotic cellwith DNA encoding the GPCR polypeptide such that the cell expresses thereceptor on its surface. The cell is then contacted with a potentialantagonist in the presence of a labeled form of a ligand, such as dATP,dAMP, or UTP. The ligand can be labeled, e.g., by radioactivity,fluorescence, or any detectable label commonly known in the art. Theamount of labeled ligand bound to the receptors is measured, e.g., bymeasuring radioactivity associated with transfected cells or membranefrom these cells. If the compound binds to the receptor, the binding oflabeled ligand to the receptor is inhibited as determined by a reductionof labeled ligand which binds to the receptors. This method is called abinding assay. Naturally, this same technique can be used to determineagonists.

[0294] In a further screening procedure, mammalian cells, for example,but not limited to, CHO, HEK 293, Xenopus Oocytes, RBL-2H3, etc., whichare transfected, are used to express the receptor of interest. The cellsare loaded with an indicator dye that produces a fluorescent signal whenbound to calcium, and the cells are contacted with a test substance anda receptor agonist, such as DATP, DAMP, or UTP. Any change influorescent signal is measured over a defined period of time using, forexample, a fluorescence spectrophotometer or a fluorescence imagingplate reader. A change in the fluorescence signal pattern generated bythe ligand indicates that a compound is a potential antagonist oragonist for the receptor.

[0295] In yet another screening procedure, mammalian cells aretransfected to express the receptor of interest, and are alsotransfected with a reporter gene construct that is coupled to activationof the receptor (for example, but not limited to luciferase orbeta-galactosidase behind an appropriate promoter). The cells arecontacted with a test substance and the receptor agonist (ligand), suchas dATP, dAMP, or UTP, and the signal produced by the reporter gene ismeasured after a defined period of time. The signal can be measuredusing a luminometer, spectrophotometer, fluorimeter, or other suchinstrument appropriate for the specific reporter construct used.Inhibition of the signal generated by the ligand indicates that acompound is a potential antagonist for the receptor.

[0296] Another screening technique for antagonists or agonists involvesintroducing RNA encoding the GPCR polypeptide into cells (or CHO, HEK293, RBL-2H3, etc.) to transiently or stably express the receptor. Thereceptor cells are then contacted with the receptor ligand, such asdATP, dAMP, or UTP, and a compound to be screened. Inhibition oractivation of the receptor is then determined by detection of a signal,such as, cAMP, calcium, proton, or other ions.

Example 7 Functional Characterization of HGPRBMY8

[0297] The putative GPCR HGPRBMY8 cDNA was PCR amplified using PFU™(Stratagene). The primers used in the PCR reaction were specific to theHGPRBMY8 polynucleotide and were ordered from Gibco BRL (5 prime primer:5′-GTCCCCAAGCTTGCACCATGACGTCCACCTGCACCAACAGCA-3′ (SEQ ID NO:38). Thefollowing 3 prime primer was used to add a Flag-tag epitope to theHGPRBMY8 polypeptide for immunocytochemistry:5′-CGGGATCCTACTTGTCGTCGTCGTCCTTGTAGTCCATAGGAAAAGTAGCAG AATCGTAGGAA-3′(SEQ ID NO:39). The product from the PCR reaction was isolated from a0.8% Agarose gel (Invitrogen) and purified using a Gel Extraction Kit™from Qiagen.

[0298] The purified product was then digested overnight along with thepcDNA3.1 Hygro™ mammalian expression vector from Invitrogen using theHindIII and BamHI restriction enzymes (New England Biolabs). Thesedigested products were then purified using the Gel Extraction Kit™ fromQiagen and subsequently ligated to the pcDNA3.1 Hygro™ expression vectorusing a DNA molar ratio of 4 parts insert: 1 vector. All DNAmodification enzymes were purchased from NEB. The ligation was incubatedovernight at 16 degrees Celsius, after which time, one microliter of themix was used to transform DH5 alpha cloning efficiency competent E.coli™ (Gibco BRL). A detailed description of the pcDNA3.1 Hygro™mammalian expression vector is available at the Invitrogen web site(www.Invitrogen.com). The plasmid DNA from the ampicillin resistantclones were isolated using the Wizard DNA Miniprep System™ from Promega.Positive clones were then confirmed and scaled up for purification usingthe Qiagen Maxiprep™ plasmid DNA purification kit.

[0299] Cell Line Generation:

[0300] The pcDNA3.1hygro vector containing the orphan HGPRBMY8 cDNA wereused to transfect CHO-NFAT/CRE (Aurora Biosciences) cells usingLipofectamine 2000™ according to the manufacturers specifications (GibcoBRL). Two days later, the cells were split 1:3 into selective media(DMEM 11056, 600 μg/ml Hygromycin, 200 μg/ml Zeocin, 10% FBS). All cellculture reagents were purchased from Gibco BRL-Invitrogen.

[0301] The CHO-NFAT/CRE and the CHO-NFAT G alpha 15 cell lines,transiently or stably transfected with the orphan HGPRBMY8 GPCR, wereanalyzed using the FACS Vantage SE™ (BD), fluorescence microscopy(Nikon), and the LJL Analyst™ (Molecular Devices). In this system,changes in real-time gene expression, as a consequence of constitutiveG-protein coupling of the orphan HGPRBMY8 GPCR, is examined by analyzingthe fluorescence emission of the transformed cells at 447 nm and 518 nm.The changes in gene expression can be visualized using Beta-Lactamase asa reporter, that, when induced by the appropriate signaling cascade,hydrolyzes an intracellularly loaded, membrane-permeant ester substrateCephalosporin-Coumarin-Fluorescein2/Acetoxymethyl (CCF2/AM™ AuroraBiosciences; Zlokarnik, et al., 1998). The CCF2/AM™ substrate is a7-hydroxycoumarin cephalosporin with a fluorescein attached through astable thioether linkage. Induced expression of the Beta-Lactamaseenzyme is readily apparent since each enzyme molecule produced iscapable of changing the fluorescence of many CCF2/AM™ substratemolecules. A schematic of this cell based system is shown below.

[0302] In summary, CCF2/AM ™ is a membrane permeant,intracellularly-trapped, fluorescent substrate with a cephalosporin corethat links a 7-hydroxycoumarin to a fluorescein. For the intactmolecule, excitation of the coumarin at 409 nm results in FluorescenceResonance Energy Transfer (FRET) to the fluorescein which emits greenlight at 518 nm. Production of active Beta-Lactamase results in cleavageof the Beta-Lactam ring, leading to disruption of FRET, and excitationof the coumarin only—thus giving rise to blue fluorescent emission at447 nm.

[0303] Fluorescent emissions were detected using a Nikon-TE300microscope equipped with an excitation filter (D405/10×-25), dichroicreflector (430 DCLP), and a barrier filter for dual DAPI/FITC (510 nM)to visually capture changes in Beta-Lactamase expression. The FACSVantage SE is equiped with a Coherent Enterprise II Argon Laser and aCoherent 302C Krypton laser. In flow cytometry, UV excitation at 351-364nm from the Argon Laser or violet excitation at 407 nm from the Kryptonlaser are used. The optical filters on the FACS Vantage SE are HQ460/50m and HQ535/40 m bandpass separated by a 490 dichroic mirror.

[0304] Prior to analyzing the fluorescent emissions from the cell linesas described above, the cells were loaded with the CCF2/AM substrate. A6× CCF2/AM loading buffer was prepared whereby 1 mM CCF2/AM (AuroraBiosciences) was dissolved in 100% DMSO (Sigma). Stock solution (12 μl)was added to 60 μl of 100 mg/ml Pluronic F127 (Sigma) in DMSO containing0.1% Acetic Acid (Sigma). This solution was added while vortexing to 1mL of Sort Buffer (PBS minus calcium and magnesium-Gibco-25 mMHEPES-Gibco-pH 7.4, 0.1% BSA). Cells were placed in serum-free media andthe 6× CCF2/AM was added to a final concentration of 1×. The cells werethen loaded at room temperature for one to two hours, and then subjectedto fluorescent emission analysis as described herein. Additional detailsrelative to the cell loading methods and/or instrument settings may befound by reference to the following publications: see Zlokarnik, et al.,1998; Whitney et al., 1998; and BD Biosciences,1999.

[0305] Immunocytochemistry:

[0306] The cell lines transfected and selected for expression ofFlag-epitope tagged orphan GPCRs were analyzed by immunocytochemistry.The cells were plated at 1×10³ in each well of a glass slide (VWR). Thecells were rinsed with PBS followed by acid fixation for 30 minutes atroom temperature using a mixture of 5% Glacial Acetic Acid/90% ethanol.The cells were then blocked in 2% BSA and 0.1%Triton in PBS, incubatedfor 2 h at room temperature or overnight at 4° C. A monoclonal FITCantibody directed against FLAG was diluted at 1:50 in blocking solutionand incubated with the cells for 2 h at room temperature. Cells werethen washed three times with 0.1% Triton in PBS for five minutes. Theslides were overlayed with mounting media dropwise with Biomedia-GelMount™ (Biomedia; Containing Anti-Quenching Agent). Cells were examinedat 10× magnification using the Nikon TE300 equiped with FITC filter (535nm).

[0307] There is strong evidence that certain GPCRs exhibit a cDNAconcentration-dependent constitutive activity through cAMP responseelement (CRE) luciferase reporters (Chen et al., 1999). In an effort todemonstrate functional coupling of HGPRBMY8 to known GPCR secondmessenger pathways, the HGPRBMY8 polypeptide was expressed at highconstitutive levels in the CHO-NFAT/CRE cell line. To this end, theHGPRBMY8 cDNA was PCR amplified and subcloned into the pcDNA3.1 hygro™mammalian expression vector as described herein. Early passageCHO-NFAT/CRE cells were then transfected with the resulting pcDNA3.1hygro™/HGPRBMY8 construct. Transfected and non-transfected CHO-NFAT/CREcells (control) were loaded with the CCF2 substrate and stimulated with10 nM PMA, 1 μM Thapsigargin (NFAT stimulator), and 10 μM Forskolin (CREstimulator) to fully activate the NFAT/CRE element. The cells were thenanalyzed for fluorescent emission by FACS.

[0308] The FACS profile demonstrates the constitutive activity ofHGPRBMY8 in the CHO-NFAT/CRE line as evidenced by the significantpopulation of cells with blue fluorescent emission at 447 nm (see FIG.12: Blue Cells). FIG. 12 further describes CHO-NFAT/CRE cell linestransfected with the pcDNA3.1 Hygro™/HGPRBMY8 mammalian expressionvector. The cells were analyzed via FACS according to their wavelengthemission at 518 nM (Channel R3-Green Cells), and 447 nM (Channel R2-BlueCells). As shown, overexpression of HGPRBMY8 results in functionalcoupling and subsequent activation of beta lactamase gene expression, asevidenced by the significant number of cells with fluorescent emissionat 447 nM relative to the non-transfected control CHO-NFAT/CRE cells(shown in FIG. 11). As expected, the NFAT/CRE response element in theuntransfected control cell line was not activated (i.e., beta lactamasenot induced), enabling the CCF2 substrate to remain intact, andresulting in the green fluorescent emission at 518 nM (see FIG. 11-GreenCells). FIG. 11 describes control CHO-NFAT/CRE (Nuclear Factor Activatorof Transcription (NFAT)/cAMP response element (CRE)) cell lines, in theabsence of the pcDNA3.1 Hygro™/HGPRBMY8 mammalian expression vectortransfection. The cells were analyzed via FACS (Fluorescent AssistedCell Sorter) according to their wavelength emission at 518 nM (ChannelR3-Green Cells), and 447 nM (Channel R2 -Blue Cells). As shown, the vastmajority of cells emit at 518 nM, with minimal emission observed at 447nM. The latter is expected since the NFAT/CRE response elements remaindormant in the absence of an activated G-protein dependent signaltransduction pathway (e.g., pathways mediated by Gq/11 or Gs coupledreceptors). As a result, the cell permeant, CCF2/AM™ (AuroraBiosciences; Zlokarnik, et al., 1998) substrate remains intact and emitslight at 518 nM.

[0309] A very low level of leaky Beta Lactamase expression wasdetectable as evidenced by the small population of cells emitting at 447nm. Analysis of a stable pool of cells transfected with HGPRBMY8revealed constitutive coupling of the cell population to the NFAT/CREresponse element, activation of Beta Lactamase and cleavage of thesubstrate (FIG. 12-Blue Cells). These results demonstrate thatoverexpression of HGPRBMY8 leads to constitutive coupling of signalingpathways known to be mediated by Gq/11 or G alpha 15/16 or Gs coupledreceptors that converge to activate either the NFAT or CRE responseelements respectively (Boss et al., 1996; Chen et al., 1999).

[0310] In an effort to further characterize the observed functionalcoupling of the HGPRBMY8 polypeptide, its ability to couple to the cAMPresponse element (CRE) independent of the NFAT response element wasexamined. To this end, HEK-CRE cell line that contained only theintegrated 3×CRE linked to the Beta-Lactamase reporter was transfectedwith the pcDNA3.1 hygro™/HGPRBMY8 construct. Analysis of thefluorescence emission from this stable pool showed that HGPRBMY8constitutively coupled to the cAMP mediated second messenger pathways(see FIG. 14 relative to FIG. 13). FIG. 14 describes FACS analysis ofHEK-CRE cell lines transfected with the pcDNA3.1 Hygro™/HGPRBMY8mammalian expression vector according to their wavelength emission at518 nM (Channel R3-Green Cells), and 447 nM (Channel R2-Blue Cells). Asshown, overexpression of HGPRBMY8 in the HEK-CRE cells resulted infunctional coupling, as evidenced by the insignificant background levelof cells with fluorescent emission at 447 nM. FIG. 13 describes HEK-CREcell lines in the absence of the pcDNA3.1 Hygro™/HGPRBMY8 mammalianexpression vector transfection. The cells were analyzed via FACS(Fluorescent Assisted Cell Sorter) according to their wavelengthemission at 518 nM (Channel R3-Green Cells), and 447 nM (Channel R2-BlueCells). As shown, the vast majority of cells emit at 518 nM, withminimal emission observed at 447 nM. The latter is expected since theCRE response elements remain dormant in the absence of an activatedG-protein dependent signal transduction pathway (e.g., pathways mediatedby Gs coupled receptors). As a result, the cell permeant, CCF2/AM™(Aurora Biosciences; Zlokarnik, et al., 1998) substrate remains intactand emits light at 518 nM.

[0311] Experiments have shown that known G coupled receptors dodemonstrate constitutive activation when overexpressed in the HEK-CREcell line. For example, direct activation of adenylate cyclase using 10μM Forskolin has been shown to activate CRE and the subsequent inductionof Beta-Lactamase in the HEK-CRE cell line (data not shown). Inconclusion, the results are consistent with HGPRBMY8 representing afunctional GPCR analogous to known Gs coupled receptors (Boss et al.,1996).

[0312] Demonstration of Cellular Expression:

[0313] HGPRBMY8 was tagged at the C-terminus using the Flag epitope andinserted into the pcDNA3.1 hygro™ expression vector, as describedherein. Immunocytochemistry of CHO-NFAT/CRE cell lines transfected withthe Flag-tagged HGPRBMY8 construct with FITC conjugated Anti Flagmonoclonal antibody demonstrated that HGPRBMY8 is indeed a cell surfacereceptor. The immunocytochemistry also confirmed expression of theHGPRBMY8 in the CHO-NFAT/CRE cell lines. Briefly, CHO-NFAT/CRE celllines were transfected with pcDNA3.1 hygro™/HGPRBMY8-Flag vector, fixedwith 70% methanol, and permeablized with 0.1% Triton×100. The cells werethen blocked with 1% Serum and incubated with a FITC conjugated AntiFlag monoclonal antibody at 1:50 dilution in PBS-Triton. The cells werethen washed several times with PBS-Triton, overlayed with mountingsolution, and fluorescent images were captured (see FIG. 15A-D). FIG. 15describes CHO-NFAT/CRE cell lines transfected with the pcDNA3.1Hygro™/HGPRBMY8-FLAG mammalian expression vector subjected toimmunocytochemistry using an FITC conjugated Anti Flag monoclonalantibody. Panel A shows the transfected CHO-NFAT/CRE cells under visualwavelengths, and panel B shows the fluorescent emission of the samecells at 530 nm after illumination with a mercury light source. The cellexpression is clearly evident in panel B, and is consistent with theHGPRBMY8 polypeptide representing a member of the GPCR family. Thecontrol cell line, non-transfected CHO-NFAT/CREcell line, exhibited nodetectable background fluorescence (FIG. 15). The HGPRBMY8-FLAG taggedexpressing CHO-NFAT/CRE line exhibited specific plasma membraneexpression as indicated (FIG. 15). These data provide clear evidencethat HGPRBMY8 is expressed in these cells and the majority of theprotein is localized to the cell surface. Cell surface localization inconsistent with HGPRBM8 representing a 7 transmembrane domain containingGPCR. Taken together, the data indicate that HGPRBMY8 is a cell surfaceGPCR that can function through increases in either cAMP or Ca²⁺ signaltransduction pathways via G alpha 15.

[0314] Screening Paradigm

[0315] The Aurora Beta-Lactamase technology provides a clear path foridentifying agonists and antagonists of the HGPRBMY8 polypeptide. Celllines that exhibit a range of constitutive coupling activity have beenidentified by sorting through HGPRBMY8 transfected cell lines using theFACS Vantage SE (see FIG. 16). For example, cell lines have been sortedthat have an intermediate level of orphan GPCR expression, which alsocorrelates with an intermediate coupling response, using the LJLanalyst. Such cell lines will provide the opportunity to screen,indirectly, for both agonists and antogonists of HGPRBMY8 by looking forinhibitors that block the beta lactamase response, or agonists thatincrease the beta lactamase response. As described herein, modulatingthe expression level of beta lactamase directly correlates with thelevel of cleaved CCF2 substrate. For example, this screening paradigmhas been shown to work for the identification of modulators of a knownGPCR, 5HT6, that couples through Adenylate Cyclase, in addition to, theidentification of modulators of the 5HT2c GPCR, that couples throughchanges in [Ca²⁺]i. The data shown below represent cell lines that havebeen engineered with the desired pattern of HGPRBMY8 expression toenable the identification of potent small molecule agonists andantagonists. HGPRBMY8 modulator screens may be carried out using avariety of high throughput methods known in the art, though preferablyusing the fully automated Aurora UHTSS system. The untransfectedCHO-NFAT/CRE cell line represents the relative background level of betalactamase expression (FIG. 16; panel a). FIG. 16 describes severalCHO-NFAT/CRE cell lines transfected with the pcDNA3.1 Hygro™/HGPRBMY8mammalian expression vector isolated via FACS that had eitherintermediate or high beta lactamase expression levels of constitutiveactivation. Panel A shows untransfected CHO-NFAT/CRE cells prior tostimulation with 10 nM PMA, 1 μM Thapsigargin, and 10 μM Forskolin(−PIT/F). Panel B shows CHO-NFAT/CRE cells after stimulation with 10 nMPMA, 1 μM Thapsigargin, and 10 μM Forskolin (+P/T/F). Panel C shows arepresentative orphan GPCR (oGPCR) transfected CHO-NFAT/CRE cells thathave an intermediate level of beta lactamase expression. Panel D shows arepresentative orphan GPCR transfected CHO-NFAT/CRE that have a highlevel of beta lactamase expression. Following treatment with a cocktailof 10 nM PMA, 1 μM Thapsigargin, and 10 μM Forskolin (FIG. 16; P/T/F;panel b), the cells fully activate the CRE-NFAT response elementdemonstrating the dynamic range of the assay. Panel C (FIG. 16)represents an orphan transfected CHO-NFAT/CRE cell line that shows anintermediate level of beta lactamase expression post P/T/F stimulation,while panel D (FIG. 16) represents a orphan transfected CHO-NFAT/CREcell line that shows a high level of constitutive beta lactamaseexpression.

Example 8 G-protein Coupled Receptor PCR Expression Profiling

[0316] RNA quantification was performed using the Taqman real-time-PCRfluorogenic assay. The Taqman assay is one of the most precise methodsfor assaying the concentration of nucleic acid templates.

[0317] All cell lines were grown using standard conditions: RPMI 1640supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100mg/ml streptomycin, and 2 mM L-glutamine, 10 mM Hepes (all fromGibcoBRL; Rockville, Md.). Eighty percent confluent cells were washedtwice with phosphate-buffered saline (GibcoBRL) and harvested using0.25% trypsin (GibcoBRL). RNA was prepared using the RNeasy Maxi Kitfrom Qiagen (Valencia, Calif.).

[0318] cDNA template for real-time PCR was generated using theSuperscript First Strand Synthesis system for RT-PCR.

[0319] SYBR Green real-time PCR reactions were prepared as follows: Thereaction mix consisted of 20 ng first strand cDNA; 50 nM Forward Primer;50 nM Reverse Primer; 0.75× SYBR Green I (Sigma); 1× SYBR Green PCRBuffer (50 mM Tris-HCl pH8.3, 75 mM KCl); 10% DMSO; 3 mM MgCl₂; 300 Meach dATP, dGTP, dTTP, dCTP; 1 U Platinum Taq DNA Polymerase HighFidelity (Cat# 11304-029; Life Technologies; Rockville, Md.); 1:50dilution; ROX (Life Technologies). Real-time PCR was performed using anApplied Biosystems 5700 Sequence Detection System. Conditions were 95Cfor 10 min (denaturation and activation of Platinum Taq DNA Polymerase),40 cycles of PCR (95C for 15 sec, 60C for 1 min). PCR products areanalyzed for uniform melting using an analysis algorithm built into the5700 Sequence Detection System.

[0320] Forward primer: 745 GPCR84-2s: 5′-GCAGAGCACTCCTCCACTCT-3′ (SEQ IDNO:34); and

[0321] Reverse primer: 746 GPCR84-2a: 5′-AGCAGGCAATCATGACAATC-3′ (SEQ IDNO:35).

[0322] cDNA quantification used in the normalization of templatequantity was performed using Taqman technology. Taqman reactions areprepared as follows: The reaction mix consisted of 20 ng first strandcDNA; 25 nM GAPDH-F3, Forward Primer; 250 nM GAPDH-R1 Reverse Primer;200 nM GAPDH-PVIC Taqman Probe (fluorescent dye labeled oligonucleotideprimer); 1× Buffer A (Applied Biosystems); 5.5 mM MgCl2; 300 M dATP,dGTP, dTTP, dCTP; 1 U Amplitaq Gold (Applied Biosystems). GAPDH,D-glyceraldehyde-3-phosphate dehydrogenase, was used as control tonormalize mRNA levels.

[0323] Real-time PCR was performed using an Applied Biosystems 7700Sequence Detection System. Conditions were 95C for 10 min. (denaturationand activation of Amplitaq Gold), 40 cycles of PCR (95C for 15 sec, 60Cfor 1 min).

[0324] The sequences for the GAPDH oligonucleotides used in the Taqmanreactions are as follows:

[0325] GAPDH-F3-5′-AGCCGAGCCACATCGCT-3′ (SEQ ID NO:60)

[0326] GAPDH-R1-5′-GTGACCAGGCGCCCAATAC-3′ (SEQ ID NO:61)

[0327] GAPDH-PVIC Taqman® Probe-VIC-5′-CAAATCCGTTGACTCCGACCTTCACCTT-3′TAMRA (SEQ ID NO:62).

[0328] The Sequence Detection System generates a Ct (threshold cycle)value that is used to calculate a concentration for each input cDNAtemplate. cDNA levels for each gene of interest are normalized to GAPDHcDNA levels to compensate for variations in total cDNA quantity in theinput sample. This is done by generating GAPDH Ct values for each cellline. Ct values for the gene of interest and GAPDH are inserted into amodified version of the Ct equation (Applied Biosystems Prism 7700Sequence Detection System User Bulletin #2), which is used to calculatea GAPDH normalized relative cDNA level for each specific cDNA. The Ctequation is as follows: relative quantity of nucleic acidtemplate=2^(Ct)=2^((Cta-Ctb)), where Cta=Ct target−Ct GAPDH, and Ctb=Ctreference−Ct GAPDH. (No reference cell line was used for the calculationof relative quantity; Ctb was defined as 21).

[0329] The Graph # of Table 1 corresponds to the tissue type positionnumber of FIG. 17. HGPRBMY8 (also known as GPCR84 or GPCR58) was foundto have relatively low expression in the tumor cell lines assayed in theOCLP-1 (oncology cell line panel). HGPRBMY8 message appears to beespecially scarce in breast tumor cell lines. The average HGPRBMY8message message level in lung tumor cell lines is 2-3 fold lower thanthe average for other cell lines assayed. TABLE 1 Ct Ct Graph NameTissue GAPDH GPCR84 dCt ddCt Quant. 1 AIN 4 breast 17.49 40 22.51 1.510.0E + 00 2 AIN 4T breast 17.15 36.8 19.65 −1.35 2.5E + 00 3 AIN4/mycbreast 17.81 40 22.19 1.19 0.0E + 00 4 BT-20 breast 17.9 36.15 18.25−2.75 6.7E + 00 5 BT-474 breast 17.65 38.34 20.69 −0.31 1.2E + 00 6BT-483 breast 17.45 35.6 18.15 −2.85 7.2E + 00 7 BT-549 breast 17.5538.21 20.66 −0.34 1.3E + 00 8 DU4475 breast 18.1 40 21.9 0.9 0.0E + 00 9H3396 breast 18.04 36.71 18.67 −2.33 5.0E + 00 10 HBL100 breast 17.0237.16 20.14 −0.86 1.8E + 00 11 Her2 MCF-7 breast 19.26 35.62 16.36 −4.642.5E + 01 12 HS 578T breast 17.83 37.28 19.45 −1.55 2.9E + 00 13 MCF7breast 17.83 40 22.17 1.17 0.0E + 00 14 MCF-7/AdrR breast 17.23 36.0118.78 −2.22 4.7E + 00 15 MDAH 2774 breast 16.87 35.24 18.37 −2.63 6.2E +00 16 MDA-MB- breast 15.72 34.08 18.36 −2.64 6.2E + 00 175-VII 17MDA-MB-231 breast 17.62 40 22.38 1.38 0.0E + 00 18 MDA-MB-453 breast17.9 37.57 19.67 −1.33 2.5E + 00 19 MDA-MB-468 breast 17.49 37.58 20.09−0.91 1.9E + 00 20 Pat-21 R60 breast 35.59 40 4.41 −16.59 ND 21 SKBR3breast 17.12 40 22.88 1.88 0.0E + 00 22 T47D breast 18.86 40 21.14 0.140.0E + 00 23 UACC-812 breast 17.06 38.26 21.2 0.2 8.7E − 01 24 ZR-75-1breast 15.95 35.36 19.41 −1.59 3.0E + 00 25 C-33A cervical 17.49 36.9619.47 −1.53 2.9E + 00 26 Ca Ski cervical 17.38 37.78 20.4 −0.6 1.5E + 0027 HeLa cervical 17.59 40 22.41 1.41 0.0E + 00 28 HT-3 cervical 17.4235.69 18.27 −2.73 6.6E + 00 29 ME-180 cervical 16.86 34.57 17.71 −3.299.8E + 00 30 SiHa cervical 18.07 40 21.93 0.93 0.0E + 00 31 SW756cervical 15.59 36.45 20.86 −0.14 1.1E + 00 32 CACO-2 colon 17.56 4022.44 1.44 0.0E + 00 33 CCD-112Co colon 18.03 40 21.97 0.97 0.0E + 00 34CCD-33Co colon 17.07 39.44 22.37 1.37 3.9E − 01 35 Colo 205 colon 18.0240 21.98 0.98 0.0E + 00 36 Colo 320DM colon 17.01 40 22.99 1.99 0.0E +00 37 Colo201 colon 17.89 34.47 16.58 −4.42 2.1E + 01 38 Cx-1 colon18.79 40 21.21 0.21 0.0E + 00 39 ddH2O colon 40 40 0 −21 ND 40 HCT116colon 17.59 36.22 18.63 −2.37 5.2E + 00 41 HCT116/epo5 colon 17.71 36.4218.71 −2.29 4.9E + 00 42 HCT116/ras colon 17.18 40 22.82 1.82 0.0E + 0043 HCT116/TX15 colon 17.36 36.91 19.55 −1.45 2.7E + 00 CR 44 HCT116/vivocolon 17.7 37.01 19.31 −1.69 3.2E + 00 45 HCT116/VM4 colon 17.87 37.5519.68 −1.32 2.5E + 00 6 46 HCT116/VP35 colon 17.3 40 22.7 1.7 0.0E + 0047 HCT-8 colon 17.44 36.86 19.42 −1.58 3.0E + 00 48 HT-29 colon 17.9 4022.1 1.1 0.0E + 00 49 LoVo colon 17.64 40 22.36 1.36 0.0E + 00 50 LS174T colon 17.93 40 22.07 1.07 0.0E + 00 51 LS123 colon 17.65 36.05 18.4−2.6 6.1E + 00 52 MIP colon 16.92 35.65 18.73 −2.27 4.8E + 00 53 SK-CO-1colon 17.75 39.84 22.09 1.09 4.7E − 01 54 SW1417 colon 17.22 39.11 21.890.89 5.4E − 01 55 SW403 colon 18.39 40 21.61 0.61 0.0E + 00 56 SW480colon 17 40 23 2 0.0E + 00 57 SW620 colon 17.16 40 22.84 1.84 0.0E + 0058 SW837 colon 18.35 37.65 19.3 −1.7 3.2E + 00 59 T84 colon 16.44 4023.56 2.56 0.0E + 00 60 CCD-18Co colon, 17.19 40 22.81 1.81 0.0E + 00fibroblast 61 HT-1080 fibrosarcoma 17.16 40 22.84 1.84 0.0E + 00 62CCRF-CEM leukemia 17.07 40 22.93 1.93 0.0E − 00 63 HL-60 leukemia 17.5440 22.46 1.46 0.0E + 00 64 K562 leukemia 18.42 40 21.58 0.58 0.0E + 0065 A-427 lung 18 40 22 1 0.0E + 00 66 A549 lung 17.63 37.06 19.43 −1.573.0E + 00 67 Calu-3 lung 18.09 37.38 19.29 −1.71 3.3E + 00 68 Calu-6lung 16.62 40 23.38 2.38 0.0E + 00 69 ChaGo-K-1 lung 17.79 37.16 19.37−1.63 3.1E + 00 70 DMS 114 lung 18.14 40 21.86 0.86 0.0E + 00 71 LX-1lung 18.17 40 21.83 0.83 0.0E + 00 72 MRC-5 lung 17.3 40 22.7 1.7 0.0E +00 73 MSTO-211H lung 16.81 40 23.19 2.19 0.0E + 00 74 NCI-H596 lung17.73 40 22.27 1.27 0.0E + 00 75 SHP-77 lung 18.66 40 21.34 0.34 0.0E +00 76 Sk-LU-1 lung 15.81 35.83 20.02 −0.98 2.0E + 00 77 SK-MES-1 lung17.1 36.33 19.23 −1.77 3.4E + 00 78 SW1271 lung 16.45 40 23.55 2.550.0E + 00 79 SW1573 lung 17.14 40 22.86 1.86 0.0E + 00 80 SW900 lung18.17 40 21.83 0.83 0.0E + 00 81 Hs 294T melanoma 17.73 35.38 17.65−3.35 1.0E + 01 82 A2780/DDP-R ovarian 21.51 40 18.49 −2.51 0.0E + 00 83A2780/DDP-S ovarian 17.89 35.73 17.84 −3.16 8.9E + 00 84 A2780/epo5ovarian 17.54 35.12 17.58 −3.42 1.1E + 01 85 A2780/TAX-R ovarian 18.438.33 19.93 −1.07 2.1E + 00 86 A2780/TAX-S ovarian 17.83 40 22.17 1.170.0E + 00 87 Caov-3 ovarian 15.5 35.35 19.85 −1.15 2.2E + 00 88 ES-2ovarian 17.22 40 22.78 1.78 0.0E + 00 89 HOC-76 ovarian 34.3 40 5.7−15.3 ND 90 OVCAR-3 ovarian 17.09 36.66 19.57 −1.43 2.7E + 00 91 PA-1ovarian 17.33 40 22.67 1.67 0.0E + 00 92 SW 626 ovarian 16.94 40 23.062.06 0.0E + 00 93 UPN251 ovarian 17.69 36.75 19.06 −1.94 3.8E + 00 94LNCAP prostate 18.17 40 21.83 0.83 0.0E + 00 95 PC-3 prostate 17.25 4022.75 1.75 0.0E + 00 96 A431 squamous 19.85 40 20.15 −0.85 0.0E + 00

Example 9 Phage Display Methods for Identifying Peptide Ligands orModulators of Orphan GPCRS

[0330] Library Construction

[0331] Two HGPRBMY libraries were used for identifying peptides that mayfunction as modulators. Specifically, a 15-mer library was used toidentify peptides that may function as agonists or antagonists. The15-mer library is an aliquot of the 15-mer library originallyconstructed by G. P. Smith (Scott, J K and Smith, G P. 1990, Science249:386-390). A 40-mer library was used for identifying natural ligandsand constructed essentially as previously described, using an M13 phagelibrary displaying random 38-amino acid peptides as a source of novelsequences with affinity to selected targets (B K Kay, et al. 1993, Gene128:59-65). This method for constructing the 40-mer library was followedwith the exception that a 15 base pair complementary region was used toanneal the two oligonucleotides, as opposed to 6, 9, or 12 base pairs,as described below.

[0332] The oligos used are: Oligo 1: 5′-CGAAGCGTAAGGGCCCAGCCGGCCNN(BNN×19) BCCGGGTCCGGGCGGC-3′ (SEQ ID NO:63) and Oligo2:5′-AAAAGGAAAAAAGCGGCCGC (VNN×20) GCCGCCCGGACCCGG-3′ (SEQ ID NO:64),where N=A+G+C+T and B=C+G+T and V=C+A+G.

[0333] The oligos were are annealed via their 15 base pair complimentarysequences which encode a constant ProGlyProGlyGly (SEQ ID NO:65)pentapeptide sequence between the random 20 amino acid segments, andthen extended by standard procedure using Klenow enzyme. This wasfollowed by endonuclease digestion using Sfi1 and Not1 enzymes andligation to Sfi1 and Not1 cleaved pCantab5E (Pharmacia). The ligationmixture was electroporated into E. coli XL1Blue and phage clones wereessentially generated as suggested by the manufacturer (Pharmacia) formaking ScFv antibody libraries in pCantab5E.

[0334] Sequencing Bound Phage

[0335] Standard procedures commonly known in the art were used. Phage ineluates were infected into E. coli host strain (TG1 for the 15-merlibrary; XL1Blue for the 40-mer library) and plated for single colonies.Colonies were grown in liquid and sequenced by standard procedure whichinvolved: 1) generating PCR product with suitable primers of the librarysegments in the phage genome (15-mer library) or pCantab5E (40-merlibrary); and 2) sequencing PCR products using one primer of each PCRprimer pair. Sequences were visually inspected or were inspected byusing the Vector NTI alignment tool.

[0336] Peptide Modulators Of The Present Invention

[0337] The following serve as non-limiting examples of HGPRBMY8 peptidemodulators: GDFWYEACESSCAFW (SEQ ID NO:66) LEWGSDVFYDVYDCC (SEQ IDNO:67) CLRSGTGCAFQLYRF (SEQ ID NO:68) NNFPCLRSGRNCDAG (SEQ ID NO:69)RIVPNGYFNVHGRSL (SEQ ID NO:70) RIDSCAKYFLRSCD (SEQ ID NO:71)

[0338] Peptide Synthesis

[0339] Peptides were synthesized on Fmoc-Knorr amide resin[N-(9-fluorenyl)methoxycarbonyl-Knorr amide-resin, Midwest Biotech,Fishers, Ind.] with an Applied Biosystems (Foster City, Calif.) model433A synthesizer and the FastMoc chemistry protocol (0.25 mmol scale)supplied with the instrument. Amino acids were double coupled as theirN-alpha-Fmoc-derivatives and reactive side chains were protected asfollows: Asp, Glu: t-Butyl ester (OtBu); Ser, Thr, Tyr: t-Butyl ether(tBu); Asn, Cys, Gln, His: Triphenylmethyl (Trt); Lys, Trp:t-Butyloxycarbonyl (Boc); Arg:2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf). After the finaldouble coupling cycle, the N-terminal Fmoc group was removed by themulti-step treatment with piperidine in N-Methylpyrrolidone as describedby the manufacturer. The N-terminal free amines were then treated with10% acetic anhydride, 5% Diisopropylamine in N-Methylpyrrolidone toyield the N-acetyl-derivative. The protected peptidyl-resins weresimultaneously deprotected and removed from the resin by standardmethods. The lyophilized peptides were purified on C₁₈ to apparenthomogeneity as judged by RP-HPLC analysis. Predicted peptide molecularweights were verified by electrospray mass spectrometry (J. Biol. Chem.vol. 273, pp.12041-12046, 1998).

[0340] Cyclic analogs were prepared from the crude linear products. Thecystine disulfide was formed using one of the following methods:

[0341] Method 1:

[0342] A sample of the crude peptide was dissolved in water at aconcentration of 0.5 mg/mL and the pH adjusted to 8.5 with NH₄OH. Thereaction was stirred at room temperature, and monitored by RP-HPLC. Oncecomplete, the reaction was brought to pH 4 with acetic acid andlyophilized. The product was purified and characterized as above.

[0343] Method 2

[0344] A sample of the crude peptide was dissolved at a concentration of0.5 mg/mL in 5% acetic acid. The pH was adjusted to 6.0 with NH₄OH. DMSO(20% by volume) was added and the reaction was stirred overnight. Afteranalytical RP-HPLC analysis, the reaction was diluted with water andtriple lyophilized to remove DMSO. The crude product was purified bypreparative RP-HPLC. (JACS. vol. 113, 6657, 1991).

[0345] Assessing Affect of Peptides on GPCR Function

[0346] The effect of any one of these peptides on the function of theGPCR of the present invention may be determined by adding an effectiveamount of each peptide to each functional assay. Representativefunctional assays are described more specifically herein, particularlyExample 7.

[0347] Uses Of The Peptide Modulators Of The Present Invention

[0348] The aforementioned peptides of the present invention are usefulfor a variety of purposes, though most notably for modulating thefunction of the GPCR of the present invention, and potentially withother GPCRs of the same G-protein coupled receptor subclass (e.g.,peptide receptors, adrenergic receptors, purinergic receptors, etc.),and/or other subclasses known in the art. For example, the peptidemodulators of the present invention may be useful as HGPRBMY8 agonists.Alternatively, the peptide modulators of the present invention may beuseful as HGPRBMY8 antagonists of the present invention. In addition,the peptide modulators of the present invention may be useful ascompetitive inhibitors of the HGPRBMY8 cognate ligand(s), or may beuseful as non-competitive inhibitors of the HGPRBMY8 cognate ligand(s).

[0349] Furthermore, the peptide modulators of the present invention maybe useful in assays designed to either deorphan the HGPRBMY8 polypeptideof the present invention, or to identify other agonists or antagonistsof the HGPRBMY8 polypeptide of the present invention, particularly smallmolecule modulators.

Example 10 Method of Creating N- and C-Terminal Deletion MutantsCorresponding to the HGPRBMY8 Polypeptide

[0350] As described elsewhere herein, the present invention encompassesthe creation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to theHGPRBMY8 polypeptide of the present invention. A number of methods areavailable to one skilled in the art for creating such mutants. Suchmethods may include a combination of PCR amplification and gene cloningmethodology. Although one of skill in the art of molecular biology,through the use of the teachings provided or referenced herein, and/orotherwise known in the art as standard methods, could readily createeach deletion mutants of the present invention, exemplary methods aredescribed below.

[0351] Briefly, using the isolated cDNA clone encoding the full-lengthHGPRBMY8 polypeptide sequence, appropriate primers of about 15-25nucleotides derived from the desired 5′ and 3′ positions of SEQ ID NO:1may be designed to PCR amplify, and subsequently clone, the intended N-and/or C-terminal deletion mutant. Such primers could comprise, forexample, an inititation and stop codon for the 5′ and 3′ primer,respectively. Such primers may also comprise restriction sites tofacilitate cloning of the deletion mutant post amplification. Moreover,the primers may comprise additional sequences, such as, for example,flag-tag sequences, kozac sequences, or other sequences discussed and/orreferenced herein.

[0352] For example, in the case of the T36 to P508 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

[0353] 5′ Primer 5′-GCAGCA GCGGCCGC ACCGTGCTGGTTATCTTCCTCGCCG-3′ (SEQ IDNO:72) NotI

[0354] 3′ Primer 5′-GCAGCA GTCGAC AGGAAAAGTAGCAGAATCGTAGG-3′ (SEQ IDNO:73) SalI

[0355] For example, in the case of the M1 to Y454 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

[0356] 5′ Primer 5′-GCAGCA GCGGCCGC ATGACGTCCACCTGCACCAACAGC-3′ (SEQ IDNO:74) NotI

[0357] 3′ Primer 5′-GCAGCA GTCGAC ATAGACATAGGGGTGGATGCAGCAC-3′ (SEQ IDNO:75) SalI

[0358] Representative PCR amplification conditions are provided below,although the skilled artisan would appreciate that other conditions maybe required for efficient amplification. A 100 μl PCR reaction mixturemay be prepared using 10 ng of the template DNA (cDNA clone ofHGPRBMY8), 200 μM 4 dNTPs, 1 μM primers, 0.25U Taq DNA polymerase (PE),and standard Taq DNA polymerase buffer. Typical PCR cycling conditionare as follows: 20-25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2min, 72 degrees 1 cycle: 10 min, 72 degrees

[0359] After the final extension step of PCR, 5U Klenow Fragment may beadded and incubated for 15 min at 30 degrees.

[0360] Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). The skilled artisan would appreciate that other plasmidscould be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E. coli cells usingmethods provided herein and/or otherwise known in the art.

[0361] The 5′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula:

(S+(X*3)) to ((S+(X*3))+25),

[0362] wherein ‘S’ is equal to the nucleotide position of the initiatingstart codon of the HGPRBMY8 gene (SEQ ID NO:1), and ‘X’ is equal to themost N-terminal amino acid of the intended N-terminal deletion mutant.The first term provides the start 5′ nucleotide position of the 5′primer, while the second term provides the end 3′ nucleotide position ofthe 5′ primer corresponding to sense strand of SEQ ID NO:1. Once thecorresponding nucleotide positions of the primer are determined, thefinal nucleotide sequence may be created by the addition of applicablerestriction site sequences to the 5′ end of the sequence, for example.As referenced herein, the addition of other sequences to the 5′ primermay be desired in certain circumstances (e.g., kozac sequences, etc.).

[0363] The 3′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula:

(S+(X*3)) to ((S+(X*3))−25),

[0364] wherein ‘S’ is equal to the nucleotide position of the initiatingstart codon of the HGPRBMY8 gene (SEQ ID NO:1), and ‘X’ is equal to themost C-terminal amino acid of the intended N-terminal deletion mutant.The first term provides the start 5′ nucleotide position of the 3′primer, while the second term provides the end 3′ nucleotide position ofthe 3′ primer corresponding to the anti-sense strand of SEQ ID NO:1.Once the corresponding nucleotide positions of the primer aredetermined, the final nucleotide sequence may be created by the additionof applicable restriction site sequences to the 5′ end of the sequence,for example. As referenced herein, the addition of other sequences tothe 3′ primer may be desired in certain circumstances (e.g., stop codonsequences, etc.). The skilled artisan would appreciate thatmodifications of the above nucleotide positions may be necessary foroptimizing PCR amplification.

[0365] The same general formulas provided above may be used inidentifying the 5′ and 3′ primer sequences for amplifying any C-terminaldeletion mutant of the present invention. Moreover, the same generalformulas provided above may be used in identifying the 5′ and 3′ primersequences for amplifying any combination of N-terminal and C-terminaldeletion mutant of the present invention. The skilled artisan wouldappreciate that modifications of the above nucleotide positions may benecessary for optimizing PCR amplification.

[0366] In preferred embodiments, the following N-terminal HGPRBMY8deletion polypeptides are encompassed by the present invention: M1-P508,T2-P508, S3-P508, T4-P508, C5-P508, T6-P508, N7-P508, S8-P508, T9-P508,R10-P508, E11-P508, S12-P508, N13-P508, S14-P508, S15-P508, H16-P508,T17-P508, C18-P508, M19-P508, P20-P508, L21-P508, S22-P508, K23-P508,M24-P508, P25-P508, I26-P508, S27-P508, L28-P508, A29-P508, H30-P508,G31-P508, I32-P508, I33-P508, R34-P508, S35-P508, T36-P508, V37-P508,L38-P508, V39-P508, I40-P508, F41-P508, L42-P508, A43-P508, A44-P508,S45-P508, F46-P508, V47-P508, G48-P508, N49-P508, I50-P508, V51-P508,L52-P508, A53-P508, L54-P508, V55-P508, L56-P508, Q57-P508, R58-P508,K59-P508, P60-P508, Q61-P508, L62-P508, L63-P508, Q64-P508, V65-P508,T66-P508, N67-P508, R68-P508, F69-P508, I70-P508, F71-P508, N72-P508,L73-P508, L74-P508, V75-P508, T76-P508, D77-P508, L78-P508, L79-P508,Q80-P508, I81-P508, S82-P508, L83-P508, V84-P508, A85-P508, P86-P508,W87-P508, V88-P508, V89-P508, A90-P508, T91-P508, S92-P508, V93-P508,P94-P508, L95-P508, F96-P508, W97-P508, P98-P508, L99-P508, N100-P508,S101-P508, H102-P508, F103-P508, C104-P508, T105-P508, A106-P508,L107-P508, V108-P508, S109-P508, L110-P508, T111-P508, H112-P508,L113-P508, F114-P508, A115-P508, F116-P508, A117-P508, S118-P508,V119-P508, N120-P508, T121-P508, I122-P508, V123-P508, L124-P508,V125-P508, S126-P508, V127-P508, D128-P508, R129-P508, Y130-P508,L131-P508, S132-P508, I133-P508, I134-P508, H135-P508, P136-P508,L137-P508, S138-P508, Y139-P508, P140-P508, S141-P508, K142-P508,M143-P508, T144-P508, Q145-P508, R146-P508, R147-P508, G148-P508,Y149-P508, L150-P508, L151-P508, L152-P508, Y153-P508, G154-P508,T155-P508, W156-P508, I157-P508, V158-P508, A159-P508, I160-P508,L161-P508, Q162-P508, S163-P508, T164-P508, P165-P508, P166-P508,L167-P508, Y168-P508, G169-P508, W170-P508, G171-P508, Q172-P508,A173-P508, A174-P508, F175-P508, D176-P508, E177-P508, R178-P508,N179-P508, A180-P508, L181-P508, C182-P508, S183-P508, M184-P508,I185-P508, W186-P508, G187-P508, A188-P508, S189-P508, P190-P508,S191-P508, Y192-P508, T193-P508, I194-P508, L195-P508, S196-P508,V197-P508, V198-P508, S199-P508, F200-P508, I201-P508, V202-P508,I203-P508, P204-P508, L205-P508, I206-P508, V207-P508, M208-P508,I209-P508, A210-P508, C211-P508, Y212-P508, S213-P508, V214-P508,V215-P508, F216-P508, C217-P508, A218-P508, A219-P508, R220-P508,R221-P508, Q222-P508, H223-P508, A224-P508, L225-P508, L226-P508,Y227-P508, N228-P508, V229-P508, K230-P508, R231-P508, H232-P508,S233-P508, L234-P508, E235-P508, V236-P508, R237-P508, V238-P508,K239-P508, D240-P508, C241-P508, V242-P508, E243-P508, N244-P508,E245-P508, D246-P508, E247-P508, E248-P508, G249-P508, A250-P508,E251-P508, K252-P508, K253-P508, E254-P508, E255-P508, F256-P508,Q257-P508, D258-P508, E259-P508, S260-P508, E261-P508, F262-P508,R263-P508, R264-P508, Q265-P508, H266-P508, E267-P508, G268-P508,E269-P508, V270-P508, K271-P508, A272-P508, K273-P508, E274-P508,G275-P508, R276-P508, M277-P508, E278-P508, A279-P508, K280-P508,D281-P508, G282-P508, S283-P508, L284-P508, K285-P508, A286-P508,K287-P508, E288-P508, G289-P508, S290-P508, T291-P508, G292-P508,T293-P508, S294-P508, E295-P508, S296-P508, S297-P508, V298-P508,E299-P508, A300-P508, R301-P508, G302-P508, S303-P508, E304-P508,E305-P508, V306-P508, R307-P508, E308-P508, S309-P508, S310-P508,T311-P508, V312-P508, A313-P508, S314-P508, D315-P508, G316-P508,S317-P508, M318-P508, E319-P508, G320-P508, K321-P508, E322-P508,G323-P508, S324-P508, T325-P508, K326-P508, V327-P508, E328-P508,E329-P508, N330-P508, S331-P508, M332-P508, K333-P508, A334-P508,D335-P508, K336-P508, G337-P508, R338-P508, T339-P508, E340-P508,V341-P508, N342-P508, Q343-P508, C344-P508, S345-P508, I346-P508,D347-P508, L348-P508, G349-P508, E350-P508, D351-P508, D352-P508,M353-P508, E354-P508, F355-P508, G356-P508, E357-P508, D358-P508,D359-P508, I360-P508, N361-P508, F362-P508, S363-P508, E364-P508,D365-P508, D366-P508, V367-P508, E368-P508, A369-P508, V370-P508,N371-P508, I372-P508, P373-P508, E374-P508, S375-P508, L376-P508,P377-P508, P378-P508, S379-P508, R380-P508, R381-P508, N382-P508,S383-P508, N384-P508, S385-P508, N386-P508, P387-P508, P388-P508,L389-P508, P390-P508, R391-P508, C392-P508, Y393-P508, Q394-P508,C395-P508, K396-P508, A397-P508, A398-P508, K399-P508, V400-P508,I401-P508, F402-P508, I403-P508, I404-P508, I405-P508, F406-P508,S407-P508, Y408-P508, V409-P508, L410-P508, S411-P508, L412-P508,G413-P508, P414-P508, Y415-P508, C416-P508, F417-P508, L418-P508,A419-P508, V420-P508, L421-P508, A422-P508, V423-P508, W424-P508,V425-P508, D426-P508, V427-P508, E428-P508, T429-P508, Q430-P508,V431-P508, P432-P508, Q433-P508, W434-P508, V435-P508, I436-P508,T437-P508, I438-P508, I439-P508, I440-P508, W441-P508, L442-P508,F443-P508, F444-P508, L445-P508, Q446-P508, C447-P508, C448-P508,I449-P508, H450-P508, P451-P508, Y452-P508, V453-P508, Y454-P508,G455-P508, Y456-P508, M457-P508, H458-P508, K459-P508, T460-P508,I461-P508, K462-P508, K463-P508, E464-P508, I465-P508, Q466-P508,D467-P508, M468-P508, L469-P508, K470-P508, K471-P508, F472-P508,F473-P508, C474-P508, K475-P508, E476-P508, K477-P508, P478-P508,P479-P508, K480-P508, E481-P508, D482-P508, S483-P508, H484-P508,P485-P508, D486-P508, L487-P508, P488-P508, G489-P508, T490-P508,E491-P508, G492-P508, G493-P508, T494-P508, E495-P508, G496-P508,K497-P508, I498-P508, V499-P508, P500-P508, S501-P508, and/or Y502-P508of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseN-terminal HGPRBMY8 deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

[0367] In preferred embodiments, the following C-terminal HGPRBMY8deletion polypeptides are encompassed by the present invention: M1-P508,M1-F507, M1-T506, M1-A505, M1-S504, M1-D503, M1-Y502, M1-S501, M1-P500,M1-V499, M1-I498, M1-K497, M1-G496, M1-E495, M1-T494, M1-G493, M1-G492,M1-E491, M1-T490, M1-G489, M1-P488, M1-L487, M1-D486, M1-P485, M1-H484,M1-S483, M1-D482, M1-E481, M1-K480, M1-P479, M1-P478, M1-K477, M1-E476,M1-K475, M1-C474, M1-F473, M1-F472, M1-K471, M1-K470, M1-L469, M1-M468,M1-D467, M1-Q466, M1-I465, M1-E464, M1-K463, M1-K462, M1-I461, M1-T460,M1-K459, M1-H458, M1-M457, M1-Y456, M1-G455, M1-Y454, M1-V453, M1-Y452,M1-P451, M1-H450, M1-I449, M1-C448, M1-C447, M1-Q446, M1-L445, M1-F444,M1-F443, M1-L442, M1-W441, M1-I440, M1-I439, M1-I438, M1-T437, M1-I436,M1-V435, M1-W434, M1-Q433, M1-P432, M1-V431, M1-Q430, M1-T429, M1-E428,M1-V427, M1-D426, M1-V425, M1-W424, M1-V423, M1-A422, M1-I421, M1-V420,M1-A419, M1-L418, M1-F417, M1-C416, M1-Y415, M1-P414, M1-G413, M1-L412,M1-S411, M1-L410, M1-V409, M1-Y408, M1-S407, M1-F406, M1-I405, M1-I404,M1-I403, M1-F402, M1-I401, M1-V400, M1-K399, M1-A398, M1-A397, M1-K396,M1-C395, M1-Q394, M1-Y393, M1-C392, M1-R391, M1-P390, M1-L389, M1-P388,M1-P387, M1-N386, M1-S385, M1-N384, M1-S383, M1-N382, M1-R381, M1-R380,M1-S379, M1-P378, M1-P377, M1-L376, M1-S375, M1-E374, M1-P373, M1-I372,M1-N371, M1-V370, M1-A369, M1-E368, M1-V367, M1-D366, M1-D365, M1-E364,M1-S363, M1-F362, M1-N361, M1-I360, M1-D359, M1-D358, M1-E357, M1-G356,M1-F355, M1-E354, M1-M353, M1-D352, M1-D351, M1-E350, M1-G349, M1-L348,M1-D347, M1-I346, M1-S345, M1-C344, M1-Q343, M1-N342, M1-V341, M1-E340,M1-T339, M1-R338, M1-G337, M1-K336, M1-D335, M1-A334, M1-K333, M1-M332,M1-S331, M1-N330, M1-E329, M1-E328, M1-V327, M1-K326, M1-T325, M1-S324,M1-G323, M1-E322, M1-K321, M1-G320, M1-E319, M1-M318, M1-S317, M1-G316,M1-D315, M1-S314, M1-A313, M1-V312, M1-T311, M1-S310, M1-S309, M1-E308,M1-R307, M1-V306, M1-E305, M1-E304, M1-S303, M1-G302, M1-R301, M1-A300,M1-E299, M1-V298, M1-S297, M1-S296, M1-E295, M1-S294, M1-T293, M1-G292,M1-T291, M1-S290, M1-G289, M1-E288, M1-K287, M1-A286, M1-K285, M1-L284,M1-S283, M1-G282, M1-D281, M1-K280, M1-A279, M1-E278, M1-M277, M1-R276,M1-G275, M1-E274, M1-K273, M1-A272, M1-K271, M1-V270, M1-E269, M1-G268,M1-E267, M1-H266, M1-Q265, M1-R264, M1-R263, M1-F262, M1-E261, M1-S260,M1-E259, M1-D258, M1-Q257, M1-F256, M1-E255, M1-E254, M1-K253, M1-K252,M1-E251, M1-A250, M1-G249, M1-E248, M1-E247, M1-D246, M1-E245, M1-N244,M1-E243, M1-V242, M1-C241, M1-D240, M1-K239, M1-V238, M1-R237, M1-V236,M1-E235, M1-L234, M1-S233, M1-H232, M1-R231, M1-K230, M1-V229, M1-N228,M1-Y227, M1-L226, M1-L225, M1-A224, M1-H223, M1-Q222, M1-R221, M1-R220,M1-A219, M1-A218, M1-C217, M1-F216, M1-V215, M1-V214, M1-S213, M1-Y212,M1-C211, M1-A210, M1-I209, M1-M208, M1-V207, M1-I206, M1-L205, M1-P204,M1-I203, M1-V202, M1-I201, M1-F200, M1-S199, M1-V198, M1-V197, M1-S 196,M1-L195, M1-I194, M1-T193, M1-Y192, M1-S191, M1-P190, M1-S189, M1-A188,M1-G187, M1-W186, M1-I185, M1-M184, M1-S183, M1-C182, M1-L181, M1-A180,M1-N179, M1-R178, M1-E177, M1-D176, M1-F175, M1-A174, M1-A173, M1-Q172,M1-G171, M1-W170, M1-G169, M1-Y168, M1-L167, M1-P166, M1-P165, M1-T164,M1-S163, M1-Q162, M1-L161, M1-I160, M1-A159, M1-V158, M1-I157, M1-W156,M1-T155, M1-G154, M1-Y153, M1-L152, M1-L151, M1-L150, M1-Y149, M1-G148,M1-R147, M1-R146, M1-Q145, M1-T144, M1-M143, M1-K142, M1-S141, M1-P140,M1-Y139, M1-S138, M1-L137, M1-P136, M1-H135, M1-I134, M1-I133, M1-S132,M1-L131, M1-Y130, M1-R129, M1-D128, M1-V127, M1-S126, M1-V125, M1-L124,M1-V123, M1-I122, M1-T121, M1-N120, M1-V119, M1-S118, M1-A117, M1-F116,M1-A115, M1-F114, M1-L113, M1-H112, M1-T111, M1-L110, M1-S109, M1-V108,M1-L107, M1-A106, M1-T105, M1-C104, M1-F103, M1-H102, M1-S101, M1-N100,M1-L99, M1-P98, M1-W97, M1-F96, M1-L95, M1-P94, M1-V93, M1-S92, M1-T91,M1-A90, M1-V89, M1-V88, M1-W87, M1-P86, M1-A85, M1-V84, M1-L83, M1-S82,M1-I81, M1-Q80, M1-L79, M1-L78, M1-D77, M1-T76, M1-V75, M1-L74, M1-L73,M1-N72, M1-F71, M1-I70, M1-F69, M1-R68, M1-N67, M1-T66, M1-V65, M1-Q64,M1-L63, M1-L62, M1-Q61, M1-P60, M1-K59, M1-R58, M1-Q57, M1-L56, M1-V55,M1-L54, M1-A53, M1-L52, M1-V51, M1-I50, M1-N49, M1-G48, M1-V47, M1-F46,M1-S45, M1-A44, M1-A43, M1-L42, M1-F41, M1-I40, M1-V39, M1-L38, M1-V37,M1-T36, M1-S35, M1-R34, M1-I33, M1-I32, M1-G31, M1-H30, M1-A29, M1-L28,M1-S27, M1-I26, M1-P25, M1-M24, M1-K23, M1-S22, M1-L21, M1-P20, M1-M19,M1-C18, M1-T17, M1-H16, M1-S15, M1-S14, M1-N13, M1-S12, M1-E11, M1-R10,M1-T9, M1-S8, and/or M1-N7 of SEQ ID NO:2. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these C-terminal HGPRBMY8 deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0368] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the HGPRBMY8 polypeptide (e.g., any combination ofboth N- and C-terminal HGPRBMY8 polypeptide deletions) of SEQ ID NO:2.For example, internal regions could be defined by the equation: aminoacid NX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of HGPRBMY8 (SEQ ID NO:2), and where CX refers toany C-terminal deletion polypeptide amino acid of HGPRBMY8 (SEQ IDNO:2). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of these polypeptides asan immunogenic and/or antigenic epitope as described elsewhere herein.

Example 11 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention Through Molecular Evolution

[0369] Although many of the most biologically active proteins known arehighly effective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, pharmaceutical, and/or industrial applications. Among thesetraits, a short physiological half-life is the most prominent problem,and is present either at the level of the protein, or the level of theproteins mRNA. The ability to extend the half-life, for example, wouldbe particularly important for a proteins use in gene therapy, transgenicanimal production, the bioprocess production and purification of theprotein, and use of the protein as a chemical modulator among others.Therefore, there is a need to identify novel variants of isolatedproteins possessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

[0370] Thus, one aspect of the present invention relates to the abilityto enhance specific characteristics of invention through directedmolecular evolution. Such an enhancement may, in a non-limiting example,benefit the inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

[0371] For example, an engineered G-protein coupled receptor may beconstitutively active upon binding of its cognate ligand. Alternatively,an engineered G-protein coupled receptor may be constitutively active inthe absence of ligand binding. In yet another example, an engineeredGPCR may be capable of being activated with less than all of theregulatory factors and/or conditions typically required for GPCRactivation (e.g., ligand binding, phosphorylation, conformationalchanges, etc.). Such GPCRs would be useful in screens to identify GPCRmodulators, among other uses described herein.

[0372] Directed evolution is comprised of several steps. The first stepis to establish a library of variants for the gene or protein ofinterest. The most important step is to then select for those variantsthat entail the activity you wish to identify. The design of the screenis essential since your screen should be selective enough to eliminatenon-useful variants, but not so stringent as to eliminate all variants.The last step is then to repeat the above steps using the best variantfrom the previous screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

[0373] Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

[0374] Random mutagenesis has been the most widely recognized method todate. Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

[0375] While both of the aforementioned methods are effective forcreating randomized pools of macromolecule variants, a third method,termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747,(1994)) has recently been elucidated. DNA shuffling has also beenreferred to as “directed molecular evolution”, “exon-shuffling”,“directed enzyme evolution”, “in vitro evolution”, and “artificialevolution”. Such reference terms are known in the art and areencompassed by the invention. This new, preferred, method apparentlyovercomes the limitations of the previous methods in that it not onlypropagates positive traits, but simultaneously eliminates negativetraits in the resulting progeny.

[0376] DNA shuffling accomplishes this task by combining the principalof in vitro recombination, along with the method of “error-prone” PCR.In effect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments—further diversifying the potential hybridization sitesduring the annealing step of the reaction.

[0377] A variety of reaction conditions could be utilized to carry-outthe DNA shuffling reaction. However, specific reaction conditions forDNA shuffling are provided, for example, in PNAS, 91:10747, (1994).Briefly, the DNA substrate to be subjected to the DNA shuffling reactionis prepared. Preparation may be in the form of simply purifying the DNAfrom contaminating cellular material, chemicals, buffers,oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entailthe use of DNA purification kits as those provided by Qiagen, Inc., orby the Promega, Corp., for example.

[0378] Once the DNA substrate has been purified, it would be subjectedto Dnase I digestion. About 2-4 μg of the DNA substrate(s) would bedigested with 0.0015 units of Dnase I (Sigma) per microliter in 100 μlof 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature.The resulting fragments of 10-50 bp could then be purified by runningthem through a 2% low-melting point agarose gel by electrophoresis ontoDE81 ion-exchange paper (Whatmann) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCl,followed by ethanol precipitation.

[0379] The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris•HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/μl. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s,and 72 C for 30 s). The referred primers would be primers correspondingto the nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

[0380] The resulting shuffled, assembled, and amplified product can bepurified using methods well known in the art (e.g., Qiagen PCRpurification kits) and then subsequently cloned using appropriaterestriction enzymes.

[0381] Although a number of variations of DNA shuffling have beenpublished to date, such variations would be obvious to the skilledartisan and are encompassed by the invention. The DNA shuffling methodcan also be tailored to the desired level of mutagenesis using themethods described by Zhao, et al. (Nucl Acid Res., 25(6):1307-1308,(1997).

[0382] As described above, once the randomized pool has been created, itcan then be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

[0383] DNA shuffling has several advantages. First, it makes use ofbeneficial mutations. When combined with screening, DNA shuffling allowsthe discovery of the best mutational combinations and does not assumethat the best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16,000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

[0384] A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

[0385] Finally, recombination enables parallel processing. Thisrepresents a significant advantage since there are likely multiplecharacteristics that would make a protein more desirable (e.g.solubility, activity, etc.). Since it is increasingly difficult toscreen for more than one desirable trait at a time, other methods ofmolecular evolution tend to be inhibitory. However, using recombination,it would be possible to combine the randomized fragments of the bestrepresentative variants for the various traits, and then select formultiple properties at once.

[0386] DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self” molecule, but rather as a “foreign”, and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

[0387] Likewise, the invention encompasses the application of DNAshuffling technology to the evolution of polynucleotides andpolypeptides of the invention, wherein one or more cycles of DNAshuffling include, in addition to the gene template DNA,oligonucleotides coding for known allelic sequences, optimized codonsequences, known variant sequences, known polynucleotide polymorphismsequences, known ortholog sequences, known homologue sequences,additional homologous sequences, additional non-homologous sequences,sequences from another species, and any number and combination of theabove.

[0388] In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

[0389] Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

Example 12 Method of Discovering Additional Single NucleotidePolymorphisms (SNPS) of the Present Invention

[0390] Additional SNPs may be discovered in the polynucleotides of thepresent invention based on comparative DNA sequencing of PCR productsderived from genomic DNA from multiple individuals. The genomic DNAsamples may be purchased from Coriell Institute (Collingswood, N.J.).PCR amplicons may be designed to cover the entire coding region of theexons using the Primer3 program (Rozen S 2000). Exon-intron structure ofcandidate genes and intron sequences may be obtained by blastn search ofGenbank cDNA sequences against the human genome draft sequences. Thesizes of these PCR amplicons will vary according to the exon-intronstructure. All the samples may be amplified from genomic DNA (20 ng) inreactions (50 μl) containing 10 mM Tris-Cl pH 8.3, 50 mM KCl, 2.5 mMMgCl₂, 150 uM dNTPs, 3 uM PCR primers, and 3.75 U TaqGold DNA polymerase(PE Biosystems).

[0391] PCR is performed in MJ Research Tetrad machines under a cyclingcondition of 94 degrees 10 min, 30 cycles of 94 degrees 30 sec, 60degrees 30 sec, and 72 degrees 30 sec, followed by 72 degrees 7 min. PCRproducts may be purified using QIAquick PCR purification kit (Qiagen),and may be sequenced by the dye-terminator method using PRISM 3700automated DNA sequencer (Applied Biosystems, Foster City, Calif.)following the manufacturer's instruction outlined in the Owner's Manual(which is hereby incorporated herein by reference in its entirety).Sequencing results may be analyzed for the presence of polymorphismsusing PolyPhred software (Nickerson D A 1997; Rieder M J 1999). All thesequence traces of potential polymorphisms may be visually inspected toconfirm the presence of SNPs.

[0392] Alternative methods for identifying SNPs of the present inventionare known in the art. One such method involves resequencing of targetsequences from individuals of diverse ethnic and geographic backgroundsby hybridization to probes immobilized to microfabricated arrays. Thestrategy and principles for the design and use of such arrays aregenerally described in WO 95/11995.

[0393] A typical probe array used in such an analysis would have twogroups of four sets of probes that respectively tile both strands of areference sequence. A first probe set comprises a plurality of probesexhibiting perfect complementarily with one of the reference sequences.Each probe in the first probe set has an interrogation position thatcorresponds to a nucleotide in the reference sequence. That is, theinterrogation position is aligned with the corresponding nucleotide inthe reference sequence, when the probe and reference sequence arealigned to maximize complementarily between the two. For each probe inthe first set, there are three corresponding probes from threeadditional probe sets. Thus, there are four probes corresponding to eachnucleotide in the reference sequence. The probes from the threeadditional probe sets would be identical to the corresponding probe fromthe first probe set except at the interrogation position, which occursin the same position in each of the four corresponding probes from thefour probe sets, and is occupied by a different nucleotide in the fourprobe sets. In the present analysis, probes may be nucleotides long.Arrays tiled for multiple different references sequences may be includedon the same substrate.

[0394] Publicly available sequences for a given gene can be assembledinto Gap4(http://www.biozentrum.unibas.ch/-biocomp/staden/Overview.html). PCRprimers covering each exon, could be designed, for example, using Primer3 (httP://www-genome.wi.mit.edu/cgi-bin/prime/primer3.cgi). Primerswould not be designed in regions where there are sequence discrepanciesbetween reads. Genomic DNA could be amplified from at least twoindividuals using 2.5 pmol each primer, 1.5 mM MgCl2, 100˜M dNTPs,0.75˜M AmpliTaq GOLD polymerase, and about 19 ng DNA in a 15 ulreaction. Reactions could be assembled using a PACKARD MultiPROBErobotic pipetting station and then put in MJ 96-well tetradthermocyclers (96° C. for minutes, followed by cycles of 96° C. forseconds, 59° C. for 2 minutes, and 72° C. for 2 minutes). A subset ofthe PCR assays for each individual could then be run on 3% NuSieve gelsin 0.5× TBE to confirm that the reaction worked.

[0395] For a given DNA, 5 ul (about 50 ng) of each PCR or RT -PCRproduct could be pooled (Final volume=150-200 ul). The products can bepurified using QiaQuick PCR purification from Qiagen. The samples wouldthen be eluted once in 35 ul sterile water and 4 ul lOX One-Phor-Allbuffer (Pharmacia). The pooled samples are then digested with 0.2 uDNaseI (Promega) for 10 minutes at 37° C. and then labeled with 0.5nmols biotin-N6-ddATP and 15 u Terminal Transferase (GibcoBRL LifeTechnology) for 60 minutes at 37° C. Both fragmentation and labelingreactions could be terminated by incubating the pooled sample for 15minutes at 100° C.

[0396] Low-density DNA chips (Affymetrix, Calif.) may be hybridizedfollowing the manufacturer's instructions. Briefly, the hybridizationcocktail consisted of 3M TMACI, mM Tris pH 7.8, 0.01% Triton X-100, 100mg/ml herring sperm DNA {Gibco BRL), 200 pM control biotin-labeledoligo. The processed PCR products are then denatured for 7 minutes at100° C. and then added to prewarmed {37° C.) hybridization solution. Thechips are hybridized overnight at 44° C. Chips are ished in 1× SSPET and6× SSPET followed by staining with 2 ug/ml SARPE and 0.5 mg/mlacetylated BSA in 200 ul of 6× SSPET for 8 minutes at room temperature.Chips are scanned using a Molecular Dynamics scanner.

[0397] Chip image files may be analyzed using Ulysses {Affymetrix,Calif.) which uses four algorithms to identify potential polymorphisms.Candidate polymorphisms may be visually inspected and assigned aconfidence value: where high confidence candidates display all threegenotypes, while likely candidates show only two genotypes {homozygousfor reference sequence and heterozygous for reference and variant). Someof the candidate polymorphisms may be confirmed by ABI sequencing.Identified polymorphisms could then be compared to several databases todetermine if they are novel.

Example 13 Method of Determining the Allele Frequency for Each SNP ofthe Present Invention

[0398] Allele frequencies of these polymorphisms may be determined bygenotyping various DNA samples (Coriell Institute; Collingswood, N.J.)using FP-TDI assay (Chen X 1999). Automated genotyping calls may be madewith an allele calling software developed by Joel Hirschorn (WhiteheadInstitute/MIT Center for Genome Research, personal communication).

[0399] Briefly, the no template controls (NTCs) may be labeledaccordingly in column C. The appropriate cells may be completed incolumn L indicating whether REF (homozygous ROX) or VAR (homozygousTAMRA) are expected to be rare genotypes (<10% of all samples)—thelatter is important in helping the program to identify rare homozygotes.The number of 96 well plates genotyped in cell P2 are noted (generallybetween 0.5 and 4)—the program works best if this is accurate. No morethan 384 samples can be analyzed at a time. The pairs of mP values fromthe LJL may be pasted into columns E and F; making sure there may be noresidual data is left at the bottom fewer than 384 data points areprovided. The DNA names may be provided in columns A, B or C; column Iwill be a concatenation of columns A, B and C. In addition, the wellnumbers for each sample may be also provided in column D.

[0400] With the above information provided, the program shouldautomatically cluster the points and identify genotypes. The programworks by converting the mP values into polar coordinates (distance fromorigin and angle from origin) with the angle being on a scale from 0 to2; heterozygotes are placed as close to 1 as possible.

[0401] The cutoff values in columns L and M may be adjusted as desired.

[0402] Expert parameters: The most important parameters are the maximumangle for REF and minimum angle for VAR. These parameters may need to bechanged in a particularly skewed assay which may be observed when an REFor VAR cluster is close to an angle of 1 and has called as a failed orHETs.

[0403] Other parameters are low and high cutoffs that are used todetermine which points are considered for the determination of edges ofthe clusters. With small numbers of data points, the high cutoff mayneed to be increased (to 500 or so). This may be the right thing to dofor every assay, but certainly when the program fails to identify asmall cluster with high signal.

[0404] NTC TAMRA and ROX indicate the position of the no templatecontrol or failed samples as estimated by the computer algorithm.

[0405] No signal=mP<is the threshold below which points areautomatically considered failures. “Throw out points with signal above”is the TAMRA or ROX mP value above which points are considered failures.The latter may occasionally need to be adjusted from 250 to 300, butcaveat emptor for assays with signals >250. ‘Lump’ or ‘split’ describesa subtle difference in the way points are grouped into clusters. Lumpgenerally is better. ‘HETs expected’ in the rare case where onlyhomozygotes of either class are expected (e.g. a study of X chromosomeSNPs in males), change this to “N”.

[0406] Notes on method of clustering: The origin is defined by the NTCsor other low signal points (the position of the origin is shown as “NTCTAMRA” and “NTC ROX”); the points with very low or high signal are notconsidered initially. The program finds the point farthest from theorigin and calls that a HET; the ROX/TAMRA ratio is calculated from thispoint, placing the heterozygotes at 45 degrees from the origin (an angleof “1”). The angles from the origin are calculated (the scale rangesfrom 0 to 2) and used to define clusters. A histogram of angles isgenerated. The cluster boundaries are defined by an algorithm that takesinto account the shape of the histogram. The homozygote clusters aredefined as the leftmost and rightmost big clusters (unless the allele isspecified as being rare, in which case the cluster need not be big). Theheterozygote is the biggest cluster in between the REF and VAR. If thereare two equal clusters, the one best-separated from REF and VAR iscalled HET. All other clusters are failed. Some fine tuning is appliedto lump in scattered points on the edges of the clusters (if “Lump” isselected). The boundaries of the clusters are “Angles” in column L.

[0407] Once the clusters are defined, the interquartile distance ofsignal intensity is defined for each cluster. Points falling more than 3or 4 interquartiles from the mean are excluded. (These are the “Signalcutoffs” in column M).

[0408] Allele frequency of the B1 receptor R317Q variant (AE103s1) is asfollows. 7% in African Americans (7/94), 0% in Caucasians (0/94), 0% inAsians (0/60), and 0% in Amerindians (0/20). Higher frequency of thisform in African Americans than in Caucasians matches the profile of apotential genetic risk factor for angioedema, which is observed morefrequently in African Americans than in Caucasians (Brown NJ 1996; BrownNJ 1998; Agostoni A 1999; Coats 2000).

[0409] The invention encompasses additional methods of determinig theallelic frequency of the SNPs of the present invention. Such methods maybe known in the art, some of which are described elsewhere herein.

Example 14 Alternative Methods of Detecting Polymorphisms Encompassed bythe Present Invention

[0410] Preparation of Samples

[0411] Polymorphisms are detected in a target nucleic acid from anindividual being analyzed. For assay of genomic DNA, virtually anybiological sample (other than pure red blood cells) is suitable. Forexample, convenient tissue samples include whole blood, semen, saliva,tears, urine, fecal material, sweat, buccal, skin and hair. For assay ofcDNA or mRNA, the tissue sample must be obtained from an organ in whichthe target nucleic acid is expressed. For example, if the target nucleicacid is a cytochrome P450, the liver is a suitable source.

[0412] Many of the methods described below require amplification of DNAfrom target samples. This can be accomplished by e.g., PCR. Seegenerally PCR Technology: Principles and Applications for DNAAmplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCRProtocols: A Guide to Methods and Applications (eds. Innis, et al.,Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic AcidsRes. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1,(1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat.No. 4,683,202.

[0413] Other suitable amplification methods include the ligase chainreaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989), Landegren etal., Science 241:1077 (1988), transcription amplification (Kwoh et al.,Proc. Natl. Acad. Sci. USA 86, 1173 (1989), and self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990))and nucleic acid based sequence amplification (NASBA). The latter twoamplification methods involve isothermal reactions based on isothermaltranscription, which produce both single stranded RNA (ssRNA) and doublestranded DNA (dsDNA) as the amplification products in a ratio of about30 or 100 to 1, respectively.

[0414] Additional methods of amplification are known in the art or aredescribed elsewhere herein.

[0415] Detection of Polymorphisms in Target DNA

[0416] There are two distinct types of analysis of target DNA fordetecting polymorphisms. The first type of analysis, sometimes referredto as de novo characterization, is carried out to identify polymorphicsites not previously characterized (i.e., to identify newpolymorphisms). This analysis compares target sequences in differentindividuals to identify points ofvariation, i.e., polymorphic sites. Byanalyzing groups of individuals representing the greatest ethnicdiversity among humans and greatest breed and species variety in plantsand animals, patterns characteristic of the most commonalleles/haplotypes of the locus can be identified, and the frequenciesof such alleles/haplotypes in the population can be determined.Additional allelic frequencies can be determined for subpopulationscharacterized by criteria such as geography, race, or gender. The denovo identification ofpolymorphisms of the invention is described in theExamples section.

[0417] The second type of analysis determines which form(s) ofacharacterized (known) polymorphism are present in individuals undertest. Additional methods of analysis are known in the art or aredescribed elsewhere herein.

[0418] Allele-Specific Probes

[0419] The design and use of allele-specific probes for analyzingpolymorphisms is described by e.g., Saiki et al., Nature 324,163-166(1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specificprobes can be designed that hybridize to a segment of target DNA fromone individual but do not hybridize to the corresponding segment fromanother individual due to the presence of different polymorphic forms inthe respective segments from the two individuals. Hybridizationconditions should be sufficiently stringent that there is a significantdifference in hybridization intensity between alleles, and preferably anessentially binary response, whereby a probe hybridizes to only one ofthe alleles. Some probes are designed to hybridize to a segment oftarget DNA such that the polymorphic site aligns with a central position(e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9position) of the probe. This design of probe achieves gooddiscrimination in hybridization between different allelic forms.

[0420] Allele-specific probes are often used in pairs, one member of apair showing a perfect match to a reference form of a target sequenceand the other member showing a perfect match to a variant form. Severalpairs of probes can then be immobilized on the same support forsimultaneous analysis of multiple polymorphisms within the same targetsequence.

[0421] Tiling Arrays

[0422] The polymorphisms can also be identified by hybridization tonucleic acid arrays, some examples of which are described in WO95/11995. The same arrays or different arrays can be used for analysisof characterized polymorphisms. WO 95/11995 also describes sub arraysthat are optimized for detection of a variant form of a precharacterizedpolymorphism. Such a subarray contains probes designed to becomplementary to a second reference sequence, which is an allelicvariant of the first reference sequence. The second group of probes isdesigned by the same principles as described, except that the probesexhibit complementarity to the second reference sequence. The inclusionof a second group (or further groups) can be particularly useful foranalyzing short subsequences of the primary reference sequence in whichmultiple mutations are expected to occur within a short distancecommensurate with the length of the probes (e.g., two or more mutationswithin 9 to bases).

[0423] Allele-Specific Primers

[0424] An allele-specific primer hybridizes to a site on target DNAoverlapping a polymorphism and only primes amplification of an allelicform to which the primer exhibits perfect complementarity. See Gibbs,Nucleic Acid Res. 17,2427-2448 (1989). This primer is used inconjunction with a second primer which hybridizes at a distal site.Amplification proceeds from the two primers, resulting in a detectableproduct which indicates the particular allelic form is present. Acontrol is usually performed with a second pair ofprimers, one ofwhichshows a single base mismatch at the polymorphic site and the other ofwhich exhibits perfect complementarity to a distal site. The single-basemismatch prevents amplification and no detectable product is formed. Themethod works best when the mismatch is included in the 3′-most positionof the oligonucleotide aligned with the polymorphism because thisposition is most destabilizing elongation from the primer (see, e.g., WO93/22456).

[0425] Direct-Sequencing

[0426] The direct analysis of the sequence of polymorphisms of thepresent invention can be accomplished using either the dideoxy chaintermination method or the Maxam-Gilbert method (see Sambrook et al.,Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989);Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

[0427] Denaturing Gradient Gel Electrophoresis

[0428] Amplification products generated using the polymerase chainreaction can be analyzed by the use of denaturing gradient gelelectrophoresis. Different alleles can be identified based on thedifferent sequence-dependent melting properties and electrophoreticmigration of DNA in solution. Erlich, ed., PCR Technology. Principlesand Applications for DNA Amplification, (W. H. Freeman and Co, New York,1992), Chapter 7.

[0429] Single-Strand Conformation Polymorphism Analysis

[0430] Alleles of target sequences can be differentiated usingsingle-strand conformation polymorphism analysis, which identifies basedifferences by alteration in electrophoretic migration of singlestranded PCR products, as described in Orita et al., Proc. Nat. Acad.Sci. 86,2766-2770 (1989). Amplified PCR products can be generated asdescribed above, and heated or otherwise denatured, to form singlestranded amplification products. Single-stranded nucleic acids mayrefold or form secondary structures which are partially dependent on thebase sequence. The different electrophoretic mobilities ofsingle-stranded amplification products can be related to base-sequencedifferences between alleles of target sequences.

[0431] Single Base Extension

[0432] An alternative method for identifying and analyzing polymorphismsis based on single-base extension (SBE) of a fluorescently-labeledprimer coupled with fluorescence resonance energy transfer (FRET)between the label of the added base and the label of the primer.Typically, the method, such as that described by Chen et al., (PNAS94:10756-61 (1997), uses a locus-specific oligonucleotide primer labeledon the 5′ terminus with 5-carboxyfluorescein (F AM). This labeled primeris designed so that the 3′ end is immediately adjacent to thepolymorphic site of interest. The labeled primer is hybridized to thelocus, and single base extension of the labeled primer is performed withfluorescently-labeled dideoxyribonucleotides (ddNTPs) in dye-terminatorsequencing fashion. An increase in fluorescence of the added ddNTP inresponse to excitation at the wavelength of the labeled primer is usedto infer the identity of the added nucleotide.

[0433] The contents of all patents, patent applications, published PCTapplications and articles, books, references, reference manuals andabstracts cited herein are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

[0434] As various changes can be made in the above-described subjectmatter without departing from the scope and spirit of the presentinvention, it is intended that all subject matter contained in the abovedescription, or defined in the appended claims, be interpreted asdescriptive and illustrative of the present invention. Manymodifications and variations of the present invention are possible inlight of the above teachings.

REFERENCES

[0435] 1. Rees, S., Brown, S., Stables, J.: “Reporter gene systems forthe study of G Protein Coupled Receptor signalling in mammalian cells”.In Milligan G. (ed.): Signal Transduction: A practical approach. Oxford:Oxford University Press, 1999: 171-221.

[0436] 2. Alam, J., Cook, J. L.: “Reporter Genes: Application to thestudy of mammalian gene transcription”. Anal. Biochem. 1990; 188:245-254.

[0437] 3. Selbie, L. A. and Hill, S. J.: “G protein-coupled receptorcross-talk: The fine-tuning of multiple receptor-signaling pathways”.TiPs. 1998; 19: 87-93.

[0438] 4. Boss, V., Talpade, D. J., and Murphy, T. J.: “Induction ofNFAT mediated transcription by Gq-coupled Receptors in lympoid andnon-lymphoid cells”. JBC. 1996; 271: 10429-10432.

[0439] 5. George, S. E., Bungay, B. J., and Naylor, L. H.: “Functionalcoupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptorsin CHO cells to a cyclic AMP-responsive luciferase reporter gene”. J.Neurochem. 1997; 69: 1278-1285.

[0440] 6. Suto, C M, Igna D M: “Selection of an optimal reporter forcell-based high throughput screening assays”. J. Biomol. Screening.1997; 2: 7-12.

[0441] 7. Zlokarnik, G., Negulescu, P. A., Knapp, T. E., More, L.,Burres, N., Feng, L., Whitney, M., Roemer, K., and Tsien, R. Y.“Quantitation of transcription and clonal selection of single livingcells with a B-Lactamase Reporter”. Science. 1998; 279: 84-88.

[0442] 8. S. Fiering et. al., Genes Dev. 4, 1823 (1990).

[0443] 9. J. Karttunen and N. Shastri, PNAS 88, 3972 (1991).

[0444] 10. Hawes, B. E., Luttrell. L. M., van Biesen, T., and Lefkowitz,R. J. (1996) JBC 271, 12133-12136.

[0445] 11. Gilman, A. G. (1987) Annul. Rev. Biochem. 56, 615-649.

[0446] 12. Maniatis et al., Cold Spring Harbor Press, 1989.

[0447] 13. Salcedo, R., Ponce, M. L., Young, H. A., Wasserman, K., Ward,J. M., Kleinman, H. K., Oppenheim, J. J., Murphy, W. J. “Humanendothelial cells express CCF2 and respond to MCP-1: direct role ofMCP-1 in angiogenesis and tumor progression”. Blood. 2000; 96 (1):34-40.

[0448] 14. Sica, A., Saccani, A., Bottazzi, B., Bernasconi, S.,Allavena, P., Gaetano, B., LaRossa, G., Scotton, C., Balkwill F.,Mantovani, A. “Defective expression of the monocyte chemotactic protein1 receptor CCF2 in macrophages associated with human ovarian carcinoma”.J. Immunology. 2000; 164: 733-8.

[0449] 15. Kypson, A., Hendrickson, S., Akhter, S., Wilson, K.,McDonald, P., Lilly, R., Dolber, P., Glower, D., Lefkowitz, R., Koch, W.“Adenovirus-mediated gene transfer of the B2 AR to donor hearts enhancescardiac function”. Gene Therapy. 1999; 6: 1298-304.

[0450] 16. Dorn, G. W., Tepe, N. M., Lorenz, J. N., Kock, W. J., Ligget,S. B. “Low and high level transgenic expression of B2AR differentiallyaffect cardiac hypertrophy and function in Galpha q-overexpressingmice”. PNAS. 1999; 96: 6400-5.

[0451] 17. J. Wess. “G protein coupled receptor: molecular mechanismsinvolved in receptor activation and selectivity of G-proteinrecognition”. FASEB. 1997; 11:346-354.

[0452] 18. Whitney, M, Rockenstein, E, Cantin, G., Knapp, T., Zlokarnik,G., Sanders, P., Durick, K., Craig, F. F., and Negulescu, P. A. “Agenome-wide functional assay of signal transduction in living mammaliancells”. Nature Biotech. 1998; 16: 1329-1333.

[0453] 19. BD Biosciences: FACS Vantage SE Training Manual. Part Number11-11020-00 Rev. A. August 1999.

[0454] 20. Chen, G., Jaywickreme, C., Way, J., Armour S., Queen K.,Watson., C., Ignar, D., Chen, W. J., Kenakin, T. “Constitutive Receptorsystems for drug discovery”. J. Pharmacol. Toxicol. Methods 1999; 42:199-206.

[0455] 21. Blahos, J., Fischer, T., Brabet, I., Stauffer, D., Rovelli,G., Bockaert, J., and Pin, J. -P. “A novel Site on the G alpha-proteinthat Rocognized Heptahelical Receptors”. J. Biol. Chem. 2001; 275, No.5, 3262-69.

[0456] 22. Offermanns, S. & Simon, M. I. “G alpha 15 and G alpha 16Couple a Wide Variety of Receptors to Phospholipase C”. J. Biol. Chem.1995; 270, No. 25, 15175-80.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 102 <210> SEQ ID NO 1<211> LENGTH: 1527 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 1 atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccacacgtgcatgccc 60 ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgtgctggttatc 120 ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgcagcgcaagccg 180 cagctgctgc aggtgaccaa ccgttttatc tttaacctcc tcgtcaccgacctgctgcag 240 atttcgctcg tggccccctg ggtggtggcc acctctgtgc ctctcttctggcccctcaac 300 agccacttct gcacggccct ggttagcctc acccacctgt tcgccttcgccagcgtcaac 360 accattgtct tggtgtcagt ggatcgctac ttgtccatca tccaccctctctcctacccg 420 tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggattgtggccatc 480 ctgcagagca ctcctccact ctacggctgg ggccaggctg cctttgatgagcgcaatgct 540 ctctgctcca tgatctgggg ggccagcccc agctacacta ttctcagcgtggtgtccttc 600 atcgtcattc cactgattgt catgattgcc tgctactccg tggtgttctgtgcagcccgg 660 aggcagcatg ctctgctgta caatgtcaag agacacagct tggaagtgcgagtcaaggac 720 tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttccaggatgagagt 780 gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaatggaagccaag 840 gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtagtgtagaggcc 900 aggggcagcg aggaggtcag agagagcagc acggtggcca gcgacggcagcatggagggt 960 aaggaaggca gcaccaaagt tgaggagaac agcatgaagg cagacaagggtcgcacagag 1020 gtcaaccagt gcagcattga cttgggtgaa gatgacatgg agtttggtgaagacgacatc 1080 aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctcccacccagtcgt 1140 cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagctgctaaagtg 1200 atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctttttagcagtc 1260 ctggccgtgt gggtggatgt cgaaacccag gtaccccagt gggtgatcaccataatcatc 1320 tggcttttct tcctgcagtg ctgcatccac ccctatgtct atggctacatgcacaagacc 1380 attaagaagg aaatccagga catgctgaag aagttcttct gcaaggaaaagcccccgaaa 1440 gaagatagcc acccagacct gcccggaaca gagggtggga ctgaaggcaagattgtccct 1500 tcctacgatt ctgctacttt tccttga 1527 <210> SEQ ID NO 2<211> LENGTH: 508 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 2 Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser SerHis 1 5 10 15 Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala HisGly Ile 20 25 30 Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser PheVal Gly 35 40 45 Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys Pro Gln LeuLeu Gln 50 55 60 Val Thr Asn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp LeuLeu Gln 65 70 75 80 Ile Ser Leu Val Ala Pro Trp Val Val Ala Thr Ser ValPro Leu Phe 85 90 95 Trp Pro Leu Asn Ser His Phe Cys Thr Ala Leu Val SerLeu Thr His 100 105 110 Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val LeuVal Ser Val Asp 115 120 125 Arg Tyr Leu Ser Ile Ile His Pro Leu Ser TyrPro Ser Lys Met Thr 130 135 140 Gln Arg Arg Gly Tyr Leu Leu Leu Tyr GlyThr Trp Ile Val Ala Ile 145 150 155 160 Leu Gln Ser Thr Pro Pro Leu TyrGly Trp Gly Gln Ala Ala Phe Asp 165 170 175 Glu Arg Asn Ala Leu Cys SerMet Ile Trp Gly Ala Ser Pro Ser Tyr 180 185 190 Thr Ile Leu Ser Val ValSer Phe Ile Val Ile Pro Leu Ile Val Met 195 200 205 Ile Ala Cys Tyr SerVal Val Phe Cys Ala Ala Arg Arg Gln His Ala 210 215 220 Leu Leu Tyr AsnVal Lys Arg His Ser Leu Glu Val Arg Val Lys Asp 225 230 235 240 Cys ValGlu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe 245 250 255 GlnAsp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala 260 265 270Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys Glu 275 280285 Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly Ser Glu 290295 300 Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly305 310 315 320 Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys AlaAsp Lys 325 330 335 Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu GlyGlu Asp Asp 340 345 350 Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser GluAsp Asp Val Glu 355 360 365 Ala Val Asn Ile Pro Glu Ser Leu Pro Pro SerArg Arg Asn Ser Asn 370 375 380 Ser Asn Pro Pro Leu Pro Arg Cys Tyr GlnCys Lys Ala Ala Lys Val 385 390 395 400 Ile Phe Ile Ile Ile Phe Ser TyrVal Leu Ser Leu Gly Pro Tyr Cys 405 410 415 Phe Leu Ala Val Leu Ala ValTrp Val Asp Val Glu Thr Gln Val Pro 420 425 430 Gln Trp Val Ile Thr IleIle Ile Trp Leu Phe Phe Leu Gln Cys Cys 435 440 445 Ile His Pro Tyr ValTyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu 450 455 460 Ile Gln Asp MetLeu Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys 465 470 475 480 Glu AspSer His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly 485 490 495 LysIle Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro 500 505 <210> SEQ ID NO 3<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 3 gcaacctgtc tcacgccctc tggctgttgc c 31 <210> SEQ ID NO 4<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 4 agttagttct aaggcaaacc tt 22 <210> SEQ ID NO 5 <211> LENGTH:33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: HGPRBMY8sense primer <400> SEQUENCE: 5 ggccgaattc gcaacctgtc tcacgccctc tgg 33<210> SEQ ID NO 6 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: HGPRBMY8 anti-sense primer <400> SEQUENCE: 6ggccgaattc ggacagttca aggtttgcct tagaac 36 <210> SEQ ID NO 7 <211>LENGTH: 490 <212> TYPE: PRT <213> ORGANISM: Gallus gallus <400>SEQUENCE: 7 Met His Asn Leu Ser Ala Gln Pro Trp Gln Ala Lys Met Ala AsnLeu 1 5 10 15 Thr Tyr Asp Asn Val Thr Leu Ser Asn Arg Ser Glu Val AlaIle Gln 20 25 30 Pro Pro Thr Asn Tyr Lys Thr Val Glu Leu Val Phe Ile AlaThr Val 35 40 45 Thr Gly Ser Leu Ser Leu Val Thr Val Val Gly Asn Ile LeuVal Met 50 55 60 Leu Ser Ile Lys Val Asn Arg Gln Leu Gln Thr Val Asn AsnTyr Phe 65 70 75 80 Leu Phe Ser Leu Ala Cys Ala Asp Leu Ile Ile Gly ValPhe Ser Met 85 90 95 Asn Leu Tyr Thr Val Tyr Ile Ile Lys Gly Tyr Trp ProLeu Gly Ala 100 105 110 Val Val Cys Asp Leu Trp Leu Ala Leu Asp Tyr ValVal Ser Asn Ala 115 120 125 Ser Val Met Asn Leu Leu Ile Ile Ser Phe AspArg Tyr Phe Cys Val 130 135 140 Thr Lys Pro Leu Thr Tyr Pro Ala Arg ArgThr Thr Lys Met Ala Gly 145 150 155 160 Leu Met Ile Ala Ala Ala Trp IleLeu Ser Phe Ile Leu Trp Ala Pro 165 170 175 Ala Ile Leu Phe Trp Gln PheIle Val Gly Lys Arg Thr Val His Glu 180 185 190 Arg Glu Cys Tyr Ile GlnPhe Leu Ser Asn Pro Ala Val Thr Phe Gly 195 200 205 Thr Ala Ile Ala AlaPhe Tyr Leu Pro Val Val Ile Met Thr Val Leu 210 215 220 Tyr Ile His IleSer Leu Ala Ser Arg Ser Arg Val Arg Arg His Lys 225 230 235 240 Pro GluSer Arg Lys Glu Arg Lys Gly Lys Ser Leu Ser Phe Phe Lys 245 250 255 AlaPro Pro Val Lys Gln Asn Asn Asn Asn Ser Pro Lys Arg Ala Val 260 265 270Glu Val Lys Glu Glu Val Arg Asn Gly Lys Val Asp Asp Gln Pro Ser 275 280285 Ala Gln Thr Glu Ala Thr Gly Gln Gln Glu Glu Lys Glu Thr Ser Asn 290295 300 Glu Ser Ser Thr Val Ser Met Thr Gln Thr Thr Lys Asp Lys Pro Thr305 310 315 320 Thr Glu Ile Leu Pro Ala Gly Gln Gly Gln Ser Pro Ala HisPro Arg 325 330 335 Val Asn Pro Thr Ser Lys Trp Ser Lys Ile Lys Ile ValThr Lys Gln 340 345 350 Thr Gly Thr Glu Ser Val Thr Ala Ile Glu Ile ValPro Ala Lys Ala 355 360 365 Gly Ala Ser Asp His Asn Ser Leu Ser Asn SerArg Pro Ala Asn Val 370 375 380 Ala Arg Lys Phe Ala Ser Ile Ala Arg SerGln Val Arg Lys Lys Arg 385 390 395 400 Gln Met Ala Ala Arg Glu Lys LysVal Thr Arg Thr Ile Phe Ala Ile 405 410 415 Leu Leu Ala Phe Ile Leu ThrTrp Thr Pro Tyr Asn Val Met Val Leu 420 425 430 Ile Asn Thr Phe Cys GluThr Cys Val Pro Glu Thr Val Trp Ser Ile 435 440 445 Gly Tyr Trp Leu CysTyr Val Asn Ser Thr Ile Asn Pro Ala Cys Tyr 450 455 460 Ala Leu Cys AsnAla Thr Phe Lys Lys Thr Phe Lys His Leu Leu Met 465 470 475 480 Cys GlnTyr Arg Asn Ile Gly Thr Ala Arg 485 490 <210> SEQ ID NO 8 <211> LENGTH:488 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400>SEQUENCE: 8 Met Cys Phe Ala Glu Lys Gly Glu Gly Ala Gly Glu Asp Val AspHis 1 5 10 15 His Ser Leu Phe Cys Pro Lys Lys Leu Val Gly Asn Leu LysGly Phe 20 25 30 Ile Arg Asn Gln Tyr His Gln His Glu Thr Ile Gln Ile LeuLys Gly 35 40 45 Ser Ala Leu Phe Leu Leu Val Leu Trp Thr Ile Phe Ala AsnSer Leu 50 55 60 Val Phe Ile Val Leu Tyr Lys Asn Pro Arg Leu Gln Thr ValPro Asn 65 70 75 80 Leu Leu Val Gly Asn Leu Ala Phe Ser Asp Leu Ala LeuGly Leu Ile 85 90 95 Val Leu Pro Leu Ser Ser Val Tyr Ala Ile Ala Gly GluTrp Val Phe 100 105 110 Pro Asp Ala Leu Cys Glu Val Phe Val Ser Ala AspIle Leu Cys Ser 115 120 125 Thr Ala Ser Ile Trp Asn Leu Ser Ile Val GlyLeu Asp Arg Tyr Trp 130 135 140 Ala Ile Thr Ser Pro Val Ala Tyr Met SerLys Arg Asn Lys Arg Thr 145 150 155 160 Ala Gly Ile Met Ile Leu Ser ValTrp Ile Ser Ser Ala Leu Ile Ser 165 170 175 Leu Ala Pro Leu Leu Gly TrpLys Gln Thr Ala Gln Thr Pro Asn Leu 180 185 190 Ile Tyr Glu Lys Asn AsnThr Val Arg Gln Cys Thr Phe Leu Asp Leu 195 200 205 Pro Ser Tyr Thr ValTyr Ser Ala Thr Gly Ser Phe Phe Ile Pro Thr 210 215 220 Leu Leu Met PhePhe Val Tyr Phe Lys Ile Tyr Gln Ala Phe Ala Lys 225 230 235 240 His ArgAla Arg Gln Ile Tyr Arg Gln Lys Val Ile Arg Lys His Ile 245 250 255 GluSer Thr Ile Leu His Glu Ile Ser His Val Leu Pro Thr Ser Asp 260 265 270Glu Phe Ala Lys Glu Glu Glu Glu Glu Glu Asp Ser Glu Ser Ser Gly 275 280285 Gln Val Glu Asn Gly Leu Gly Asn Gly Asn Asp Ala Ile Ile Glu Glu 290295 300 Asp Glu Cys Glu Asp Glu Asp Ser Asp Glu Lys Arg Asp Asp His Thr305 310 315 320 Ser Met Thr Thr Val Thr Ala Thr Val Thr Gly Pro Thr GluAla Pro 325 330 335 Tyr Met Lys Arg Glu Ala Lys Ile Ser Lys Ser Val ProIle Glu Lys 340 345 350 Glu Ser Ala Ile Gln Lys Arg Glu Ala Lys Pro MetArg Ser Val Met 355 360 365 Ala Ile Ser Tyr Glu Lys Val Lys Arg His LysAsn Arg Lys Glu Arg 370 375 380 Ile Tyr Arg Lys Ser Leu Gln Arg Lys ProLys Ala Ile Ser Ala Ala 385 390 395 400 Lys Glu Arg Arg Gly Val Lys ValLeu Gly Ile Ile Leu Gly Cys Phe 405 410 415 Thr Val Cys Trp Ala Pro PhePhe Thr Met Tyr Val Leu Val Gln Phe 420 425 430 Cys Lys Asp Cys Ser ProAsn Ala His Ile Glu Met Phe Ile Thr Trp 435 440 445 Leu Gly Tyr Ser AsnSer Ala Met Asn Pro Ile Ile Tyr Thr Val Phe 450 455 460 Asn Arg Asp TyrGln Ile Ala Leu Lys Arg Leu Phe Thr Ser Glu Lys 465 470 475 480 Lys ProSer Ser Thr Ser Arg Val 485 <210> SEQ ID NO 9 <211> LENGTH: 423 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9 Met Asp Leu ArgAla Thr Ser Ser Asn Asp Ser Asn Ala Thr Ser Gly 1 5 10 15 Tyr Ser AspThr Ala Ala Val Asp Trp Asp Glu Gly Glu Asn Ala Thr 20 25 30 Gly Ser GlySer Leu Pro Asp Pro Glu Leu Ser Tyr Gln Ile Ile Thr 35 40 45 Ser Leu PheLeu Gly Ala Leu Ile Leu Cys Ser Ile Phe Gly Asn Ser 50 55 60 Cys Val ValAla Ala Ile Ala Leu Glu Arg Ser Leu Gln Asn Val Ala 65 70 75 80 Asn TyrLeu Ile Gly Ser Leu Ala Val Thr Asp Leu Met Val Ser Val 85 90 95 Leu ValLeu Pro Met Ala Ala Leu Tyr Gln Val Leu Asn Lys Trp Thr 100 105 110 LeuGly Gln Asp Ile Cys Asp Leu Phe Ile Ala Leu Asp Val Leu Cys 115 120 125Cys Thr Ser Ser Ile Leu His Leu Cys Ala Ile Ala Leu Asp Arg Tyr 130 135140 Trp Ala Ile Thr Asp Pro Ile Asp Tyr Val Asn Lys Arg Thr Pro Arg 145150 155 160 Arg Ala Ala Val Leu Ile Ser Val Thr Trp Leu Ile Gly Phe SerIle 165 170 175 Ser Ile Pro Pro Met Leu Gly Trp Arg Ser Ala Glu Asp ArgAla Asn 180 185 190 Pro Asp Ala Cys Ile Ile Ser Gln Asp Pro Gly Tyr ThrIle Tyr Ser 195 200 205 Thr Phe Gly Ala Phe Tyr Ile Pro Leu Ile Leu MetLeu Val Leu Tyr 210 215 220 Gly Arg Ile Phe Lys Ala Ala Arg Phe Arg IleArg Lys Thr Val Lys 225 230 235 240 Lys Thr Glu Lys Ala Lys Ala Ser AspMet Cys Leu Thr Leu Ser Pro 245 250 255 Ala Val Phe His Lys Arg Ala AsnGly Asp Ala Val Ser Ala Glu Trp 260 265 270 Lys Arg Gly Tyr Lys Phe LysPro Ser Ser Pro Cys Ala Asn Gly Ala 275 280 285 Val Arg His Gly Glu GluMet Glu Ser Leu Glu Ile Ile Glu Val Asn 290 295 300 Ser Asn Ser Lys ThrHis Leu Pro Leu Pro Asn Thr Pro Gln Ser Ser 305 310 315 320 Ser His GluAsn Ile Asn Glu Lys Thr Thr Gly Thr Arg Arg Lys Ile 325 330 335 Ala LeuAla Arg Glu Arg Lys Thr Val Lys Thr Leu Gly Ile Ile Met 340 345 350 GlyThr Phe Ile Phe Cys Trp Leu Pro Phe Phe Ile Val Ala Leu Val 355 360 365Leu Pro Phe Cys Ala Glu Asn Cys Tyr Met Pro Glu Trp Leu Gly Ala 370 375380 Val Ile Asn Trp Leu Gly Tyr Ser Asn Ser Leu Leu Asn Pro Ile Ile 385390 395 400 Tyr Ala Tyr Phe Asn Lys Asp Phe Gln Ser Ala Phe Lys Lys IleLeu 405 410 415 Arg Cys Lys Phe His Arg His 420 <210> SEQ ID NO 10 <211>LENGTH: 421 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:10 Met Asp Met Phe Ser Leu Gly Gln Gly Asn Asn Thr Thr Thr Ser Leu 1 510 15 Glu Pro Phe Gly Thr Gly Gly Asn Asp Thr Gly Leu Ser Asn Val Thr 2025 30 Phe Ser Tyr Gln Val Ile Thr Ser Leu Leu Leu Gly Thr Leu Ile Phe 3540 45 Cys Ala Val Leu Gly Asn Ala Cys Val Val Ala Ala Ile Ala Leu Glu 5055 60 Arg Ser Leu Gln Asn Val Ala Asn Tyr Leu Ile Gly Ser Leu Ala Val 6570 75 80 Thr Asp Leu Met Val Ser Val Leu Val Leu Pro Met Ala Ala Leu Tyr85 90 95 Gln Val Leu Asn Lys Trp Thr Leu Gly Gln Val Thr Cys Asp Leu Phe100 105 110 Ile Ala Leu Asp Val Leu Cys Cys Thr Ser Ser Ile Leu His LeuCys 115 120 125 Ala Ile Ala Leu Asp Arg Tyr Trp Ala Ile Thr Asp Pro IleAsp Tyr 130 135 140 Val Asn Lys Arg Thr Pro Arg Arg Ala Ala Ala Leu IleSer Leu Thr 145 150 155 160 Trp Leu Ile Gly Phe Leu Ile Ser Ile Pro ProMet Leu Gly Trp Arg 165 170 175 Ala Pro Glu Asp Arg Ser Asn Pro Asn GluCys Thr Ile Ser Lys Asp 180 185 190 His Gly Tyr Thr Ile Tyr Ser Thr PheGly Ala Phe Tyr Ile Pro Leu 195 200 205 Leu Leu Met Leu Val Leu Tyr GlyArg Ile Phe Arg Ala Ala Arg Phe 210 215 220 Arg Ile Arg Lys Thr Val LysLys Val Glu Lys Lys Gly Ala Gly Thr 225 230 235 240 Ser Phe Gly Thr SerSer Ala Pro Pro Pro Lys Lys Ser Leu Asn Gly 245 250 255 Gln Pro Gly SerGly Asp Cys Arg Arg Ser Ala Glu Asn Arg Ala Val 260 265 270 Gly Thr ProCys Ala Asn Gly Ala Val Arg Gln Gly Glu Asp Asp Ala 275 280 285 Thr LeuGlu Val Ile Glu Val His Arg Val Gly Asn Ser Lys Gly Asp 290 295 300 LeuPro Leu Pro Ser Glu Ser Gly Ala Thr Ser Tyr Val Pro Ala Cys 305 310 315320 Leu Glu Arg Lys Asn Glu Arg Thr Ala Glu Ala Lys Arg Lys Met Ala 325330 335 Leu Ala Arg Glu Arg Lys Thr Val Lys Thr Leu Gly Ile Ile Met Gly340 345 350 Thr Phe Ile Leu Cys Trp Leu Pro Phe Phe Ile Val Ala Leu ValLeu 355 360 365 Pro Phe Cys Glu Ser Ser Cys His Met Pro Glu Leu Leu GlyAla Ile 370 375 380 Ile Asn Trp Leu Gly Tyr Ser Asn Ser Leu Leu Asn ProVal Ile Tyr 385 390 395 400 Ala Tyr Phe Asn Lys Asp Phe Gln Asn Ala PheLys Lys Ile Ile Lys 405 410 415 Cys Lys Phe Cys Arg 420 <210> SEQ ID NO11 <211> LENGTH: 423 <212> TYPE: PRT <213> ORGANISM: Fugu rubripes <400>SEQUENCE: 11 Met Asp Leu Arg Ala Thr Ser Ser Asn Asp Ser Asn Ala Thr SerGly 1 5 10 15 Tyr Ser Asp Thr Ala Ala Val Asp Trp Asp Glu Gly Glu AsnAla Thr 20 25 30 Gly Ser Gly Ser Leu Pro Asp Pro Glu Leu Ser Tyr Gln IleIle Thr 35 40 45 Ser Leu Phe Leu Gly Ala Leu Ile Leu Cys Ser Ile Phe GlyAsn Ser 50 55 60 Cys Val Val Ala Ala Ile Ala Leu Glu Arg Ser Leu Gln AsnVal Ala 65 70 75 80 Asn Tyr Leu Ile Gly Ser Leu Ala Val Thr Asp Leu MetVal Ser Val 85 90 95 Leu Val Leu Pro Met Ala Ala Leu Tyr Gln Val Leu AsnLys Trp Thr 100 105 110 Leu Gly Gln Asp Ile Cys Asp Leu Phe Ile Ala LeuAsp Val Leu Cys 115 120 125 Cys Thr Ser Ser Ile Leu His Leu Cys Ala IleAla Leu Asp Arg Tyr 130 135 140 Trp Ala Ile Thr Asp Pro Ile Asp Tyr ValAsn Lys Arg Thr Pro Arg 145 150 155 160 Arg Ala Ala Val Leu Ile Ser ValThr Trp Leu Ile Gly Phe Ser Ile 165 170 175 Ser Ile Pro Pro Met Leu GlyTrp Arg Ser Ala Glu Asp Arg Ala Asn 180 185 190 Pro Asp Ala Cys Ile IleSer Gln Asp Pro Gly Tyr Thr Ile Tyr Ser 195 200 205 Thr Phe Gly Ala PheTyr Ile Pro Leu Ile Leu Met Leu Val Leu Tyr 210 215 220 Gly Arg Ile PheLys Ala Ala Arg Phe Arg Ile Arg Lys Thr Val Lys 225 230 235 240 Lys ThrGlu Lys Ala Lys Ala Ser Asp Met Cys Leu Thr Leu Ser Pro 245 250 255 AlaVal Phe His Lys Arg Ala Asn Gly Asp Ala Val Ser Ala Glu Trp 260 265 270Lys Arg Gly Tyr Lys Phe Lys Pro Ser Ser Pro Cys Ala Asn Gly Ala 275 280285 Val Arg His Gly Glu Glu Met Glu Ser Leu Glu Ile Ile Glu Val Asn 290295 300 Ser Asn Ser Lys Thr His Leu Pro Leu Pro Asn Thr Pro Gln Ser Ser305 310 315 320 Ser His Glu Asn Ile Asn Glu Lys Thr Thr Gly Thr Arg ArgLys Ile 325 330 335 Ala Leu Ala Arg Glu Arg Lys Thr Val Lys Thr Leu GlyIle Ile Met 340 345 350 Gly Thr Phe Ile Phe Cys Trp Leu Pro Phe Phe IleVal Ala Leu Val 355 360 365 Leu Pro Phe Cys Ala Glu Asn Cys Tyr Met ProGlu Trp Leu Gly Ala 370 375 380 Val Ile Asn Trp Leu Gly Tyr Ser Asn SerLeu Leu Asn Pro Ile Ile 385 390 395 400 Tyr Ala Tyr Phe Asn Lys Asp PheGln Ser Ala Phe Lys Lys Ile Leu 405 410 415 Arg Cys Lys Phe His Arg His420 <210> SEQ ID NO 12 <211> LENGTH: 509 <212> TYPE: PRT <213> ORGANISM:Lymnaea stagnalis <400> SEQUENCE: 12 Met Ala Asn Phe Thr Phe Gly Asp LeuAla Leu Asp Val Ala Arg Met 1 5 10 15 Gly Gly Leu Ala Ser Thr Pro SerGly Leu Arg Ser Thr Gly Leu Thr 20 25 30 Thr Pro Gly Leu Ser Pro Thr GlyLeu Val Thr Ser Asp Phe Asn Asp 35 40 45 Ser Tyr Gly Leu Thr Gly Gln PheIle Asn Gly Ser His Ser Ser Arg 50 55 60 Ser Arg Asp Asn Ala Ser Ala AsnAsp Thr Ser Ala Thr Asn Met Thr 65 70 75 80 Asp Asp Arg Tyr Trp Ser LeuThr Val Tyr Ser His Glu His Leu Val 85 90 95 Leu Thr Ser Val Ile Leu GlyLeu Phe Val Leu Cys Cys Ile Ile Gly 100 105 110 Asn Cys Phe Val Ile AlaAla Val Met Leu Glu Arg Ser Leu His Asn 115 120 125 Val Ala Asn Tyr LeuIle Leu Ser Leu Ala Val Ala Asp Leu Met Val 130 135 140 Ala Val Leu ValMet Pro Leu Ser Val Val Ser Glu Ile Ser Lys Val 145 150 155 160 Trp PheLeu His Ser Glu Val Cys Asp Met Trp Ile Ser Val Asp Val 165 170 175 LeuCys Cys Thr Ala Ser Ile Leu His Leu Val Ala Ile Ala Met Asp 180 185 190Arg Tyr Trp Ala Val Thr Ser Ile Asp Tyr Ile Arg Arg Arg Ser Ala 195 200205 Arg Arg Ile Leu Leu Met Ile Met Val Val Trp Ile Val Ala Leu Phe 210215 220 Ile Ser Ile Pro Pro Leu Phe Gly Trp Arg Asp Pro Asn Asn Asp Pro225 230 235 240 Asp Lys Thr Gly Thr Cys Ile Ile Ser Gln Asp Lys Gly TyrThr Ile 245 250 255 Phe Ser Thr Val Gly Ala Phe Tyr Leu Pro Met Leu ValMet Met Ile 260 265 270 Ile Tyr Ile Arg Ile Trp Leu Val Ala Arg Ser ArgIle Arg Lys Asp 275 280 285 Lys Phe Gln Met Thr Lys Ala Arg Leu Lys ThrGlu Glu Thr Thr Leu 290 295 300 Val Ala Ser Pro Lys Thr Glu Tyr Ser ValVal Ser Asp Cys Asn Gly 305 310 315 320 Cys Asn Ser Pro Asp Ser Thr ThrGlu Lys Lys Lys Arg Arg Ala Pro 325 330 335 Phe Lys Ser Tyr Gly Cys SerPro Arg Pro Glu Arg Lys Lys Asn Arg 340 345 350 Ala Lys Lys Leu Pro GluAsn Ala Asn Gly Val Asn Ser Asn Ser Ser 355 360 365 Ser Ser Glu Arg LeuLys Gln Ile Gln Ile Glu Thr Ala Glu Ala Phe 370 375 380 Ala Asn Gly CysAla Glu Glu Ala Ser Ile Ala Met Leu Glu Arg Gln 385 390 395 400 Cys AsnAsn Gly Lys Lys Ile Ser Ser Asn Asp Thr Pro Tyr Ser Arg 405 410 415 ThrArg Glu Lys Leu Glu Leu Lys Arg Glu Arg Lys Ala Ala Arg Thr 420 425 430Leu Ala Ile Ile Thr Gly Ala Phe Leu Ile Cys Trp Leu Pro Phe Phe 435 440445 Ile Ile Ala Leu Ile Gly Pro Phe Val Asp Pro Glu Gly Ile Pro Pro 450455 460 Phe Ala Arg Ser Phe Val Leu Trp Leu Gly Tyr Phe Asn Ser Leu Leu465 470 475 480 Asn Pro Ile Ile Tyr Thr Ile Phe Ser Pro Glu Phe Arg SerAla Phe 485 490 495 Gln Lys Ile Leu Phe Gly Lys Tyr Arg Arg Gly His Arg500 505 <210> SEQ ID NO 13 <211> LENGTH: 572 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 13 Met Thr Phe Arg Asp Leu LeuSer Val Ser Phe Glu Gly Pro Arg Pro 1 5 10 15 Asp Ser Ser Ala Gly GlySer Ser Ala Gly Gly Gly Gly Gly Ser Ala 20 25 30 Gly Gly Ala Ala Pro SerGlu Gly Pro Ala Val Gly Gly Val Pro Gly 35 40 45 Gly Ala Gly Gly Gly GlyGly Val Val Gly Ala Gly Ser Gly Glu Asp 50 55 60 Asn Arg Ser Ser Ala GlyGlu Pro Gly Ser Ala Gly Ala Gly Gly Asp 65 70 75 80 Val Asn Gly Thr AlaAla Val Gly Gly Leu Val Val Ser Ala Gln Gly 85 90 95 Val Gly Val Gly ValPhe Leu Ala Ala Phe Ile Leu Met Ala Val Ala 100 105 110 Gly Asn Leu LeuVal Ile Leu Ser Val Ala Cys Asn Arg His Leu Gln 115 120 125 Thr Val ThrAsn Tyr Phe Ile Val Asn Leu Ala Val Ala Asp Leu Leu 130 135 140 Leu SerAla Thr Val Leu Pro Phe Ser Ala Thr Met Glu Val Leu Gly 145 150 155 160Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp Val Trp Ala Ala Val Asp 165 170175 Val Leu Cys Cys Thr Ala Ser Ile Leu Ser Leu Cys Thr Ile Ser Val 180185 190 Asp Arg Tyr Val Gly Val Arg His Ser Leu Lys Tyr Pro Ala Ile Met195 200 205 Thr Glu Arg Lys Ala Ala Ala Ile Leu Ala Leu Leu Trp Val ValAla 210 215 220 Leu Val Val Ser Val Gly Pro Leu Leu Gly Trp Lys Glu ProVal Pro 225 230 235 240 Pro Asp Glu Arg Phe Cys Gly Ile Thr Glu Glu AlaGly Tyr Ala Val 245 250 255 Phe Ser Ser Val Cys Ser Phe Tyr Leu Pro MetAla Val Ile Val Val 260 265 270 Met Tyr Cys Arg Val Tyr Val Val Ala ArgSer Thr Thr Arg Ser Leu 275 280 285 Glu Ala Gly Val Lys Arg Glu Arg GlyLys Ala Ser Glu Val Val Leu 290 295 300 Arg Ile His Cys Arg Gly Ala AlaThr Gly Ala Asp Gly Ala His Gly 305 310 315 320 Met Arg Ser Ala Lys GlyHis Thr Phe Arg Ser Ser Leu Ser Val Arg 325 330 335 Leu Leu Lys Phe SerArg Glu Lys Lys Ala Ala Lys Thr Leu Ala Ile 340 345 350 Val Val Gly ValPhe Val Leu Cys Trp Phe Pro Phe Phe Phe Val Leu 355 360 365 Pro Leu GlySer Leu Phe Pro Gln Leu Lys Pro Ser Glu Gly Val Phe 370 375 380 Lys ValIle Phe Trp Leu Gly Tyr Phe Asn Ser Cys Val Asn Pro Leu 385 390 395 400Ile Tyr Pro Cys Ser Ser Arg Glu Phe Lys Arg Ala Phe Leu Arg Leu 405 410415 Leu Arg Cys Gln Cys Arg Arg Arg Arg Arg Arg Arg Pro Leu Trp Arg 420425 430 Val Tyr Gly His His Trp Arg Ala Ser Thr Ser Gly Leu Arg Gln Asp435 440 445 Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro Gly Ala Pro Leu AlaLeu 450 455 460 Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro Pro Gly Thr ProGlu Met 465 470 475 480 Gln Ala Pro Val Ala Ser Arg Arg Lys Pro Pro SerAla Phe Arg Glu 485 490 495 Trp Arg Leu Leu Gly Pro Phe Arg Arg Pro ThrThr Gln Leu Arg Ala 500 505 510 Lys Val Ser Ser Leu Ser His Lys Ile ArgAla Gly Gly Ala Gln Arg 515 520 525 Ala Glu Ala Ala Cys Ala Gln Arg SerGlu Val Glu Ala Val Ser Leu 530 535 540 Gly Val Pro His Glu Val Ala GluGly Ala Thr Cys Gln Ala Tyr Glu 545 550 555 560 Leu Ala Asp Tyr Ser AsnLeu Arg Glu Thr Asp Ile 565 570 <210> SEQ ID NO 14 <211> LENGTH: 562<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 14 Met ThrPhe Arg Asp Ile Leu Ser Val Thr Phe Glu Gly Pro Arg Ala 1 5 10 15 SerSer Ser Thr Gly Gly Ser Gly Ala Gly Gly Gly Ala Gly Thr Val 20 25 30 GlyPro Glu Gly Pro Ala Val Gly Gly Val Pro Gly Ala Thr Gly Gly 35 40 45 SerAla Val Val Gly Thr Gly Ser Gly Glu Asp Asn Gln Ser Ser Thr 50 55 60 AlaGlu Ala Gly Ala Ala Ala Ser Gly Glu Val Asn Gly Ser Ala Ala 65 70 75 80Val Gly Gly Leu Val Val Ser Ala Gln Gly Val Gly Val Gly Val Phe 85 90 95Leu Ala Ala Phe Ile Leu Thr Ala Val Ala Gly Asn Leu Leu Val Ile 100 105110 Leu Ser Val Ala Cys Asn Arg His Leu Gln Thr Val Thr Asn Tyr Phe 115120 125 Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Ser Ala Ala Val Leu130 135 140 Pro Phe Ser Ala Thr Met Glu Val Leu Gly Phe Trp Pro Phe GlyArg 145 150 155 160 Thr Phe Cys Asp Val Trp Ala Ala Val Asp Val Leu CysCys Thr Ala 165 170 175 Ser Ile Leu Ser Leu Cys Thr Ile Ser Val Asp ArgTyr Val Gly Val 180 185 190 Arg His Ser Leu Lys Tyr Pro Ala Ile Met ThrGlu Arg Lys Ala Ala 195 200 205 Ala Ile Leu Ala Leu Leu Trp Ala Val AlaLeu Val Val Ser Val Gly 210 215 220 Pro Leu Leu Gly Trp Lys Glu Pro ValPro Pro Asp Glu Arg Phe Cys 225 230 235 240 Gly Ile Thr Glu Glu Val GlyTyr Ala Ile Phe Ser Ser Val Cys Ser 245 250 255 Phe Tyr Leu Pro Met AlaVal Ile Val Val Met Tyr Cys Arg Val Tyr 260 265 270 Val Val Ala Arg SerThr Thr Arg Ser Leu Glu Ala Gly Ile Lys Arg 275 280 285 Glu Pro Gly LysAla Ser Glu Val Val Leu Arg Ile His Cys Arg Gly 290 295 300 Ala Ala ThrSer Ala Lys Gly Asn Pro Gly Thr Gln Ser Ser Lys Gly 305 310 315 320 HisThr Leu Arg Ser Ser Leu Ser Val Arg Leu Leu Lys Phe Ser Arg 325 330 335Glu Lys Lys Ala Ala Lys Thr Leu Ala Ile Val Val Gly Val Phe Val 340 345350 Leu Cys Trp Phe Pro Phe Phe Phe Val Leu Pro Leu Gly Ser Leu Phe 355360 365 Pro Gln Leu Lys Pro Ser Glu Gly Val Phe Lys Val Ile Phe Trp Leu370 375 380 Gly Tyr Phe Asn Ser Cys Val Asn Pro Leu Ile Tyr Pro Cys SerSer 385 390 395 400 Arg Glu Phe Lys Arg Ala Phe Leu Arg Leu Leu Arg CysGln Cys Arg 405 410 415 Arg Arg Arg Arg Arg Leu Trp Pro Ser Leu Arg ProPro Leu Ala Ser 420 425 430 Leu Asp Arg Arg Pro Ala Leu Arg Leu Cys ProGln Pro Ala His Arg 435 440 445 Thr Pro Arg Gly Ser Pro Ser Pro His CysThr Pro Arg Pro Gly Leu 450 455 460 Arg Arg His Ala Gly Gly Ala Gly PheGly Leu Arg Pro Ser Lys Ala 465 470 475 480 Ser Leu Arg Leu Arg Glu TrpArg Leu Leu Gly Pro Leu Gln Arg Pro 485 490 495 Thr Thr Gln Leu Arg AlaLys Val Ser Ser Leu Ser His Lys Phe Arg 500 505 510 Ser Gly Gly Ala ArgArg Ala Glu Thr Ala Cys Ala Leu Arg Ser Glu 515 520 525 Val Glu Ala ValSer Leu Asn Val Pro Gln Asp Gly Ala Glu Ala Val 530 535 540 Ile Cys GlnAla Tyr Glu Pro Gly Asp Leu Ser Asn Leu Arg Glu Thr 545 550 555 560 AspIle <210> SEQ ID NO 15 <211> LENGTH: 499 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 15 Met Val Phe Leu Ser Gly Asn Ala Ser AspSer Ser Asn Cys Thr Gln 1 5 10 15 Pro Pro Ala Pro Val Asn Ile Ser LysAla Ile Leu Leu Gly Val Ile 20 25 30 Leu Gly Gly Leu Ile Leu Phe Gly ValLeu Gly Asn Ile Leu Val Ile 35 40 45 Leu Ser Val Ala Cys His Arg His LeuHis Ser Val Thr His Tyr Tyr 50 55 60 Ile Val Asn Leu Ala Val Ala Asp LeuLeu Leu Thr Ser Thr Val Leu 65 70 75 80 Pro Phe Ser Ala Ile Phe Glu ValLeu Gly Tyr Trp Ala Phe Gly Arg 85 90 95 Val Phe Cys Asn Ile Trp Ala AlaVal Asp Val Leu Cys Cys Thr Ala 100 105 110 Ser Ile Met Gly Leu Cys IleIle Ser Ile Asp Arg Tyr Ile Gly Val 115 120 125 Ser Tyr Pro Leu Arg TyrPro Thr Ile Val Thr Gln Arg Arg Gly Leu 130 135 140 Met Ala Leu Leu CysVal Trp Ala Leu Ser Leu Val Ile Ser Ile Gly 145 150 155 160 Pro Leu PheGly Trp Arg Gln Pro Ala Pro Glu Asp Glu Thr Ile Cys 165 170 175 Gln IleAsn Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser 180 185 190 PheTyr Leu Pro Leu Ala Ile Ile Leu Val Met Tyr Cys Arg Val Tyr 195 200 205Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr 210 215220 Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn 225230 235 240 Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr Lys ThrHis 245 250 255 Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys AlaAla Lys 260 265 270 Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys TrpLeu Pro Phe 275 280 285 Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro AspPhe Lys Pro Ser 290 295 300 Glu Thr Val Phe Lys Ile Val Phe Trp Leu GlyTyr Leu Asn Ser Cys 305 310 315 320 Ile Asn Pro Ile Ile Tyr Pro Cys SerSer Gln Glu Phe Lys Lys Ala 325 330 335 Phe Gln Asn Val Leu Arg Ile GlnCys Leu Arg Arg Lys Gln Ser Ser 340 345 350 Lys His Ala Leu Gly Tyr ThrLeu His Pro Pro Ser Gln Ala Val Glu 355 360 365 Gly Gln His Lys Asp MetVal Arg Ile Pro Val Gly Ser Arg Glu Thr 370 375 380 Phe Tyr Arg Ile SerLys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe 385 390 395 400 Ser Ser MetPro Arg Gly Ser Ala Arg Ile Thr Val Ser Lys Asp Gln 405 410 415 Ser SerCys Thr Thr Ala Arg Thr Lys Ser Arg Ser Val Thr Arg Leu 420 425 430 GluCys Ser Gly Met Ile Leu Ala His Cys Asn Leu Arg Leu Pro Gly 435 440 445Ser Arg Asp Ser Pro Ala Ser Ala Ser Gln Ala Ala Gly Thr Thr Gly 450 455460 Asp Val Pro Pro Gly Arg Arg His Gln Ala Gln Leu Ile Phe Val Phe 465470 475 480 Leu Val Glu Thr Gly Phe His His Val Gly Gln Asp Asp Leu AspLeu 485 490 495 Leu Thr Ser <210> SEQ ID NO 16 <211> LENGTH: 429 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 Met Val PheLeu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gln 1 5 10 15 Pro ProAla Pro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile 20 25 30 Leu GlyGly Leu Ile Leu Phe Gly Val Leu Gly Asn Ile Leu Val Ile 35 40 45 Leu SerVal Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr 50 55 60 Ile ValAsn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu 65 70 75 80 ProPhe Ser Ala Ile Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95 ValPhe Cys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110Ser Ile Met Gly Leu Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val 115 120125 Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu 130135 140 Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile Gly145 150 155 160 Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp Glu ThrIle Cys 165 170 175 Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu Phe Ser AlaLeu Gly Ser 180 185 190 Phe Tyr Leu Pro Leu Ala Ile Ile Leu Val Met TyrCys Arg Val Tyr 195 200 205 Val Val Ala Lys Arg Glu Ser Arg Gly Leu LysSer Gly Leu Lys Thr 210 215 220 Asp Lys Ser Asp Ser Glu Gln Val Thr LeuArg Ile His Arg Lys Asn 225 230 235 240 Ala Pro Ala Gly Gly Ser Gly MetAla Ser Ala Lys Thr Lys Thr His 245 250 255 Phe Ser Val Arg Leu Leu LysPhe Ser Arg Glu Lys Lys Ala Ala Lys 260 265 270 Thr Leu Gly Ile Val ValGly Cys Phe Val Leu Cys Trp Leu Pro Phe 275 280 285 Phe Leu Val Met ProIle Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser 290 295 300 Glu Thr Val PheLys Ile Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys 305 310 315 320 Ile AsnPro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala 325 330 335 PheGln Asn Val Leu Arg Ile Gln Cys Leu Arg Arg Lys Gln Ser Ser 340 345 350Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu 355 360365 Gly Gln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Arg Glu Thr 370375 380 Phe Tyr Arg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe385 390 395 400 Ser Ser Met Pro Arg Gly Ser Ala Arg Ile Thr Val Ser LysAsp Gln 405 410 415 Ser Ser Cys Thr Thr Ala Arg Gly His Thr Pro Met Thr420 425 <210> SEQ ID NO 17 <211> LENGTH: 455 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 17 Met Val Phe Leu Ser Gly AsnAla Ser Asp Ser Ser Asn Cys Thr Gln 1 5 10 15 Pro Pro Ala Pro Val AsnIle Ser Lys Ala Ile Leu Leu Gly Val Ile 20 25 30 Leu Gly Gly Leu Ile LeuPhe Gly Val Leu Gly Asn Ile Leu Val Ile 35 40 45 Leu Ser Val Ala Cys HisArg His Leu His Ser Val Thr His Tyr Tyr 50 55 60 Ile Val Asn Leu Ala ValAla Asp Leu Leu Leu Thr Ser Thr Val Leu 65 70 75 80 Pro Phe Ser Ala IlePhe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95 Val Phe Cys Asn IleTrp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110 Ser Ile Met GlyLeu Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val 115 120 125 Ser Tyr ProLeu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu 130 135 140 Met AlaLeu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile Gly 145 150 155 160Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp Glu Thr Ile Cys 165 170175 Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser 180185 190 Phe Tyr Leu Pro Leu Ala Ile Ile Leu Val Met Tyr Cys Arg Val Tyr195 200 205 Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu LysThr 210 215 220 Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His ArgLys Asn 225 230 235 240 Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala LysThr Lys Thr His 245 250 255 Phe Ser Val Arg Leu Leu Lys Phe Ser Arg GluLys Lys Ala Ala Lys 260 265 270 Thr Leu Gly Ile Val Val Gly Cys Phe ValLeu Cys Trp Leu Pro Phe 275 280 285 Phe Leu Val Met Pro Ile Gly Ser PhePhe Pro Asp Phe Lys Pro Ser 290 295 300 Glu Thr Val Phe Lys Ile Val PheTrp Leu Gly Tyr Leu Asn Ser Cys 305 310 315 320 Ile Asn Pro Ile Ile TyrPro Cys Ser Ser Gln Glu Phe Lys Lys Ala 325 330 335 Phe Gln Asn Val LeuArg Ile Gln Cys Leu Cys Arg Lys Gln Ser Ser 340 345 350 Lys His Ala LeuGly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu 355 360 365 Gly Gln HisLys Asp Met Val Arg Ile Pro Val Gly Ser Arg Glu Thr 370 375 380 Phe TyrArg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe 385 390 395 400Ser Ser Met Pro Arg Gly Ser Ala Arg Ile Thr Val Ser Lys Asp Gln 405 410415 Ser Ser Cys Thr Thr Ala Arg Arg Gly Met Asp Cys Arg Tyr Phe Thr 420425 430 Lys Asn Cys Arg Glu His Ile Lys His Val Asn Phe Met Met Pro Pro435 440 445 Trp Arg Lys Gly Leu Glu Cys 450 455 <210> SEQ ID NO 18 <211>LENGTH: 466 <212> TYPE: PRT <213> ORGANISM: Rattus norvegicus <400>SEQUENCE: 18 Met Val Leu Leu Ser Glu Asn Ala Ser Glu Gly Ser Asn Cys ThrHis 1 5 10 15 Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leu GlyVal Ile 20 25 30 Leu Gly Gly Leu Ile Ile Phe Gly Val Leu Gly Asn Ile LeuVal Ile 35 40 45 Leu Ser Val Ala Cys His Arg His Leu His Ser Val Thr HisTyr Tyr 50 55 60 Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser ThrVal Leu 65 70 75 80 Pro Phe Ser Ala Ile Phe Glu Ile Leu Gly Tyr Trp AlaPhe Gly Arg 85 90 95 Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu CysCys Thr Ala 100 105 110 Ser Ile Met Gly Leu Cys Ile Ile Ser Ile Asp ArgTyr Ile Gly Val 115 120 125 Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val ThrGln Arg Arg Gly Val 130 135 140 Arg Ala Leu Leu Cys Val Trp Val Leu SerLeu Val Ile Ser Ile Gly 145 150 155 160 Pro Leu Phe Gly Trp Arg Gln ProAla Pro Glu Asp Glu Thr Ile Cys 165 170 175 Gln Ile Asn Glu Glu Pro GlyTyr Val Leu Phe Ser Ala Leu Gly Ser 180 185 190 Phe Tyr Val Pro Leu AlaIle Ile Leu Val Met Tyr Cys Arg Val Tyr 195 200 205 Val Val Ala Lys ArgGlu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr 210 215 220 Asp Lys Ser AspSer Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn 225 230 235 240 Val ProAla Glu Gly Gly Gly Val Ser Ser Ala Lys Asn Lys Thr His 245 250 255 PheSer Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys 260 265 270Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe 275 280285 Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser 290295 300 Glu Thr Val Phe Lys Ile Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys305 310 315 320 Ile Asn Pro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe LysLys Ala 325 330 335 Phe Gln Asn Val Leu Arg Ile Gln Cys Leu Arg Arg ArgGln Ser Ser 340 345 350 Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro SerGln Ala Leu Glu 355 360 365 Gly Gln His Arg Asp Met Val Arg Ile Pro ValGly Ser Gly Glu Thr 370 375 380 Phe Tyr Lys Ile Ser Lys Thr Asp Gly ValCys Glu Trp Lys Phe Phe 385 390 395 400 Ser Ser Met Pro Gln Gly Ser AlaArg Ile Thr Val Pro Lys Asp Gln 405 410 415 Ser Ala Cys Thr Thr Ala ArgVal Arg Ser Lys Ser Phe Leu Gln Val 420 425 430 Cys Cys Cys Val Gly SerSer Ala Pro Arg Pro Glu Glu Asn His Gln 435 440 445 Val Pro Thr Ile LysIle His Thr Ile Ser Leu Gly Glu Asn Gly Glu 450 455 460 Glu Val 465<210> SEQ ID NO 19 <211> LENGTH: 466 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 19 Met Val Leu Leu Ser Glu Asn Ala Ser Glu GlySer Asn Cys Thr His 1 5 10 15 Pro Pro Ala Gln Val Asn Ile Ser Lys AlaIle Leu Leu Gly Val Ile 20 25 30 Leu Gly Gly Leu Ile Ile Phe Gly Val LeuGly Asn Ile Leu Val Ile 35 40 45 Leu Ser Val Ala Cys His Arg His Leu HisSer Val Thr His Tyr Tyr 50 55 60 Ile Val Asn Leu Ala Val Ala Asp Leu LeuLeu Thr Ser Thr Val Leu 65 70 75 80 Pro Phe Ser Ala Ile Phe Glu Ile LeuGly Tyr Trp Ala Phe Gly Arg 85 90 95 Val Phe Cys Asn Ile Trp Ala Ala ValAsp Val Leu Cys Cys Thr Ala 100 105 110 Ser Ile Met Gly Leu Cys Ile IleSer Ile Asp Arg Tyr Ile Gly Val 115 120 125 Ser Tyr Pro Leu Arg Tyr ProThr Ile Val Thr Gln Arg Arg Gly Val 130 135 140 Arg Ala Leu Leu Cys ValTrp Ala Leu Ser Leu Val Ile Ser Ile Gly 145 150 155 160 Pro Leu Phe GlyTrp Arg Gln Gln Ala Pro Glu Asp Glu Thr Ile Cys 165 170 175 Gln Ile AsnGlu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser 180 185 190 Phe TyrVal Pro Leu Thr Ile Ile Leu Val Met Tyr Cys Arg Val Tyr 195 200 205 ValVal Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr 210 215 220Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn 225 230235 240 Val Pro Ala Glu Gly Ser Gly Val Ser Ser Ala Lys Asn Lys Thr His245 250 255 Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala AlaLys 260 265 270 Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp LeuPro Phe 275 280 285 Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro Asn PheLys Pro Pro 290 295 300 Glu Thr Val Phe Lys Ile Val Phe Trp Leu Gly TyrLeu Asn Ser Cys 305 310 315 320 Ile Asn Pro Ile Ile Tyr Pro Cys Ser SerGln Glu Phe Lys Lys Ala 325 330 335 Phe Gln Asn Val Leu Arg Ile Gln CysLeu Arg Arg Arg Gln Ser Ser 340 345 350 Lys His Ala Leu Gly Tyr Thr LeuHis Pro Pro Ser Gln Ala Val Glu 355 360 365 Glu Gln His Arg Gly Met ValArg Ile Pro Val Gly Ser Gly Glu Thr 370 375 380 Phe Tyr Lys Ile Ser LysThr Asp Gly Val Cys Glu Trp Lys Phe Phe 385 390 395 400 Ser Ser Met ProGln Gly Ser Ala Arg Ile Thr Met Pro Lys Asp Gln 405 410 415 Ser Ala CysThr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gln Val 420 425 430 Cys CysCys Val Gly Ser Ser Thr Pro Arg Pro Glu Glu Asn His Gln 435 440 445 ValPro Thr Ile Lys Ile His Thr Ile Ser Leu Gly Glu Asn Gly Glu 450 455 460Glu Val 465 <210> SEQ ID NO 20 <211> LENGTH: 466 <212> TYPE: PRT <213>ORGANISM: Bos taurus <400> SEQUENCE: 20 Met Val Phe Leu Ser Gly Asn AlaSer Asp Ser Ser Asn Cys Thr His 1 5 10 15 Pro Pro Pro Pro Val Asn IleSer Lys Ala Ile Leu Leu Gly Val Ile 20 25 30 Leu Gly Gly Leu Ile Leu PheGly Val Leu Gly Asn Ile Leu Val Ile 35 40 45 Leu Ser Val Ala Cys His ArgHis Leu His Ser Val Thr His Tyr Tyr 50 55 60 Ile Val Asn Leu Ala Val AlaAsp Leu Leu Leu Thr Ser Thr Val Leu 65 70 75 80 Pro Phe Ser Ala Ile PheGlu Ile Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95 Val Phe Cys Asn Val TrpAla Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110 Ser Ile Met Gly LeuCys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val 115 120 125 Ser Tyr Pro LeuArg Tyr Pro Thr Ile Val Thr Gln Lys Arg Gly Leu 130 135 140 Met Ala LeuLeu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile Gly 145 150 155 160 ProLeu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp Glu Thr Ile Cys 165 170 175Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser 180 185190 Phe Tyr Val Pro Leu Thr Ile Ile Leu Val Met Tyr Cys Arg Val Tyr 195200 205 Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr210 215 220 Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg Ile His Arg LysAsn 225 230 235 240 Ala Gln Val Gly Gly Ser Gly Val Thr Ser Ala Lys AsnLys Thr His 245 250 255 Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu LysLys Ala Ala Lys 260 265 270 Thr Leu Gly Ile Val Val Gly Cys Phe Val LeuCys Trp Leu Pro Phe 275 280 285 Phe Leu Val Met Pro Ile Gly Ser Phe PhePro Asp Phe Arg Pro Ser 290 295 300 Glu Thr Val Phe Lys Ile Ala Phe TrpLeu Gly Tyr Leu Asn Ser Cys 305 310 315 320 Ile Asn Pro Ile Ile Tyr ProCys Ser Ser Gln Glu Phe Lys Lys Ala 325 330 335 Phe Gln Asn Val Leu ArgIle Gln Cys Leu Arg Arg Lys Gln Ser Ser 340 345 350 Lys His Thr Leu GlyTyr Thr Leu His Ala Pro Ser His Val Leu Glu 355 360 365 Gly Gln His LysAsp Leu Val Arg Ile Pro Val Gly Ser Ala Glu Thr 370 375 380 Phe Tyr LysIle Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Ile Phe 385 390 395 400 SerSer Leu Pro Arg Gly Ser Ala Arg Met Ala Val Ala Arg Asp Pro 405 410 415Ser Ala Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gln Val 420 425430 Cys Cys Cys Leu Gly Pro Ser Thr Pro Ser His Gly Glu Asn His Gln 435440 445 Ile Pro Thr Ile Lys Ile His Thr Ile Ser Leu Ser Glu Asn Gly Glu450 455 460 Glu Val 465 <210> SEQ ID NO 21 <211> LENGTH: 295 <212> TYPE:PRT <213> ORGANISM: Canis familiaris <400> SEQUENCE: 21 Met Val Phe LeuSer Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr His 1 5 10 15 Pro Pro AlaPro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile 20 25 30 Leu Gly GlyLeu Ile Ile Phe Gly Val Leu Gly Asn Ile Leu Val Ile 35 40 45 Leu Ser ValAla Cys His Arg His Leu His Ser Val Thr His Tyr Tyr 50 55 60 Ile Val AsnLeu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu 65 70 75 80 Pro PheSer Ala Ile Phe Glu Ile Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95 Val PheCys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110 SerIle Met Gly Leu Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val 115 120 125Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Lys Arg Gly Leu 130 135140 Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile Gly 145150 155 160 Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp Glu Thr IleCys 165 170 175 Gln Ile Thr Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala LeuGly Ser 180 185 190 Phe Tyr Val Pro Leu Thr Ile Ile Leu Val Met Tyr CysArg Val Tyr 195 200 205 Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys SerGly Leu Lys Thr 210 215 220 Asp Lys Ser Asp Ser Glu Gln Val Thr Leu ArgIle His Arg Lys Asn 225 230 235 240 Ala Pro Val Gly Gly Thr Gly Val SerSer Ala Lys Asn Lys Thr His 245 250 255 Phe Ser Val Arg Leu Leu Lys PheSer Arg Glu Lys Lys Ala Ala Lys 260 265 270 Thr Leu Gly Ile Val Val GlyCys Phe Val Leu Cys Trp Leu Pro Phe 275 280 285 Phe Leu Val Met Pro IleGly 290 295 <210> SEQ ID NO 22 <211> LENGTH: 466 <212> TYPE: PRT <213>ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 22 Met Val Phe Leu SerGly Asn Ala Ser Asp Ser Ser Asn Cys Thr His 1 5 10 15 Pro Pro Ala ProVal Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile 20 25 30 Leu Gly Gly LeuIle Leu Phe Gly Val Leu Gly Asn Ile Leu Val Ile 35 40 45 Leu Ser Val AlaCys His Arg His Leu His Ser Val Thr His Tyr Tyr 50 55 60 Ile Val Asn LeuAla Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu 65 70 75 80 Pro Phe SerAla Ile Phe Glu Ile Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95 Val Phe CysAsn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110 Ser IleIle Ser Leu Cys Val Ile Ser Ile Asp Arg Tyr Ile Gly Val 115 120 125 SerTyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu 130 135 140Arg Ala Leu Leu Cys Val Trp Ala Phe Ser Leu Val Ile Ser Val Gly 145 150155 160 Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Asp Asp Glu Thr Ile Cys165 170 175 Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu GlySer 180 185 190 Phe Tyr Val Pro Leu Thr Ile Ile Leu Ala Met Tyr Cys ArgVal Tyr 195 200 205 Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser GlyLeu Lys Thr 210 215 220 Asp Lys Ser Asp Ser Glu Gln Val Thr Leu Arg IleHis Arg Lys Asn 225 230 235 240 Ala Pro Ala Gly Gly Ser Gly Val Ala SerAla Lys Asn Lys Thr His 245 250 255 Phe Ser Val Arg Leu Leu Lys Phe SerArg Glu Lys Lys Ala Ala Lys 260 265 270 Thr Leu Gly Ile Val Val Gly CysPhe Val Leu Cys Trp Leu Pro Phe 275 280 285 Phe Leu Val Met Pro Ile GlySer Phe Phe Pro Asp Phe Lys Pro Pro 290 295 300 Glu Thr Val Phe Lys IleVal Phe Trp Leu Gly Tyr Leu Asn Ser Cys 305 310 315 320 Ile Asn Pro IleIle Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala 325 330 335 Phe Gln AsnVal Leu Lys Ile Gln Cys Leu Arg Arg Lys Gln Ser Ser 340 345 350 Lys HisAla Leu Gly Tyr Thr Leu His Ala Pro Ser Gln Ala Leu Glu 355 360 365 GlyGln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Gly Glu Thr 370 375 380Phe Tyr Lys Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe 385 390395 400 Ser Ser Met Pro Arg Gly Ser Ala Arg Ile Thr Val Pro Lys Asp Gln405 410 415 Ser Ala Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu GlnVal 420 425 430 Cys Cys Cys Val Gly Pro Ser Thr Pro Asn Pro Gly Glu AsnHis Gln 435 440 445 Val Pro Thr Ile Lys Ile His Thr Ile Ser Leu Ser GluAsn Gly Glu 450 455 460 Glu Val 465 <210> SEQ ID NO 23 <211> LENGTH: 466<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 23 Met ValPhe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gln 1 5 10 15 ProPro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile 20 25 30 LeuGly Gly Leu Ile Leu Phe Gly Val Leu Gly Asn Ile Leu Val Ile 35 40 45 LeuSer Val Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr 50 55 60 IleVal Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu 65 70 75 80Pro Phe Ser Ala Ile Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105110 Ser Ile Met Gly Leu Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val 115120 125 Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu130 135 140 Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser IleGly 145 150 155 160 Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp GluThr Ile Cys 165 170 175 Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu Phe SerAla Leu Gly Ser 180 185 190 Phe Tyr Leu Pro Leu Ala Ile Ile Leu Val MetTyr Cys Arg Val Tyr 195 200 205 Val Val Ala Lys Arg Glu Ser Arg Gly LeuLys Ser Gly Leu Lys Thr 210 215 220 Asp Lys Ser Asp Ser Glu Gln Val ThrLeu Arg Ile His Arg Lys Asn 225 230 235 240 Ala Pro Ala Gly Gly Ser GlyMet Ala Ser Ala Lys Thr Lys Thr His 245 250 255 Phe Ser Val Arg Leu LeuLys Phe Ser Arg Glu Lys Lys Ala Ala Lys 260 265 270 Thr Leu Gly Ile ValVal Gly Cys Phe Val Leu Cys Trp Leu Pro Phe 275 280 285 Phe Leu Val MetPro Ile Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser 290 295 300 Glu Thr ValPhe Lys Ile Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys 305 310 315 320 IleAsn Pro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala 325 330 335Phe Gln Asn Val Leu Arg Ile Gln Cys Leu Cys Arg Lys Gln Ser Ser 340 345350 Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu 355360 365 Gly Gln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Arg Glu Thr370 375 380 Phe Tyr Arg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys PhePhe 385 390 395 400 Ser Ser Met Pro Arg Gly Ser Ala Arg Ile Thr Val SerLys Asp Gln 405 410 415 Ser Ser Cys Thr Thr Ala Arg Val Arg Ser Lys SerPhe Leu Gln Val 420 425 430 Cys Cys Cys Val Gly Pro Ser Thr Pro Ser LeuAsp Lys Asn His Gln 435 440 445 Val Pro Thr Ile Lys Val His Thr Ile SerLeu Ser Glu Asn Gly Glu 450 455 460 Glu Val 465 <210> SEQ ID NO 24 <211>LENGTH: 470 <212> TYPE: PRT <213> ORGANISM: Oryzias latipes <400>SEQUENCE: 24 Met Thr Pro Ser Ser Val Thr Leu Asn Cys Ser Asn Cys Ser HisVal 1 5 10 15 Leu Ala Pro Glu Leu Asn Thr Val Lys Ala Val Val Leu GlyMet Val 20 25 30 Leu Gly Ile Phe Ile Leu Phe Gly Val Ile Gly Asn Ile LeuVal Ile 35 40 45 Leu Ser Val Val Cys His Arg His Leu Gln Thr Val Thr TyrTyr Phe 50 55 60 Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Ser Ser ThrVal Leu 65 70 75 80 Pro Phe Ser Ala Ile Phe Glu Ile Leu Asp Arg Trp ValPhe Gly Arg 85 90 95 Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu CysCys Thr Ala 100 105 110 Ser Ile Met Ser Leu Cys Val Ile Ser Val Asp ArgTyr Ile Gly Val 115 120 125 Ser Tyr Pro Leu Arg Tyr Pro Ala Ile Met ThrLys Arg Arg Ala Leu 130 135 140 Leu Ala Val Met Leu Leu Trp Val Leu SerVal Ile Ile Ser Ile Gly 145 150 155 160 Pro Leu Phe Gly Trp Lys Glu ProAla Pro Glu Asp Glu Thr Val Cys 165 170 175 Lys Ile Thr Glu Glu Pro GlyTyr Ala Ile Phe Ser Ala Val Gly Ser 180 185 190 Phe Tyr Leu Pro Leu AlaIle Ile Leu Ala Met Tyr Cys Arg Val Tyr 195 200 205 Val Val Ala Gln LysGlu Ser Arg Gly Leu Lys Glu Gly Gln Lys Ile 210 215 220 Glu Lys Ser AspSer Glu Gln Val Ile Leu Arg Met His Arg Gly Asn 225 230 235 240 Thr ThrVal Ser Glu Asp Glu Ala Leu Arg Ser Arg Thr His Phe Ala 245 250 255 LeuArg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu 260 265 270Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe Phe Leu 275 280285 Val Leu Pro Ile Gly Ser Ile Phe Pro Ala Tyr Arg Pro Ser Asp Thr 290295 300 Val Phe Lys Ile Thr Phe Trp Leu Gly Tyr Phe Asn Ser Cys Ile Asn305 310 315 320 Pro Ile Ile Tyr Leu Cys Ser Asn Gln Glu Phe Lys Lys AlaPhe Gln 325 330 335 Ser Leu Leu Gly Val His Cys Leu Arg Met Thr Pro ArgAla His His 340 345 350 His His Leu Ser Val Gly Gln Ser Gln Thr Gln GlyHis Ser Leu Thr 355 360 365 Ile Ser Leu Asp Ser Lys Gly Ala Pro Cys ArgLeu Ser Pro Ser Ser 370 375 380 Ser Val Ala Leu Ser Arg Thr Pro Ser SerArg Asp Ser Arg Glu Trp 385 390 395 400 Arg Val Phe Ser Gly Gly Pro IleAsn Ser Gly Pro Gly Pro Thr Glu 405 410 415 Ala Gly Arg Ala Lys Val AlaLys Leu Cys Asn Lys Ser Leu His Arg 420 425 430 Thr Cys Cys Cys Ile LeuArg Ala Arg Thr Pro Thr Gln Asp Pro Ala 435 440 445 Pro Leu Gly Asp LeuPro Thr Ile Lys Ile His Gln Leu Ser Leu Ser 450 455 460 Glu Lys Gly GluSer Val 465 470 <210> SEQ ID NO 25 <211> LENGTH: 391 <212> TYPE: PRT<213> ORGANISM: Branchiostoma lanceolatum <400> SEQUENCE: 25 Met Ser AlaAsn Thr Thr Val Ser Pro Thr Glu Thr Thr Ala Asn Leu 1 5 10 15 Thr AlaAsn Ser Thr Glu Ala Ser Val Gly Ser Cys Phe Ala Pro Asn 20 25 30 Pro TyrSer Ala Gly Val Gln Ala Val Leu Gly Leu Ile Thr Val Ile 35 40 45 Leu IleLeu Leu Thr Val Ile Gly Asn Val Leu Val Ile Leu Ala Val 50 55 60 Thr CysHis Arg Lys Met Arg Thr Val Thr Asn Phe Phe Ile Val Ser 65 70 75 80 LeuAla Cys Ala Asp Leu Ser Val Gly Ile Thr Val Leu Pro Phe Ala 85 90 95 AlaThr Asn Asp Ile Leu Gly Tyr Trp Pro Phe Gly Gly Tyr Cys Asp 100 105 110Val Trp Val Ser Phe Asp Val Leu Asn Ser Thr Ala Ser Ile Leu Asn 115 120125 Leu Val Val Ile Ala Phe Asp Arg Phe Leu Ala Ile Thr Ala Pro Phe 130135 140 Thr Tyr His Thr Arg Met Thr Glu Arg Thr Ala Gly Ile Leu Ile Ala145 150 155 160 Thr Val Trp Gly Ile Ser Leu Val Val Ser Phe Leu Pro IleGln Ala 165 170 175 Gly Trp Tyr Arg Asp Asn Gln Ser Glu Glu Ala Leu AlaIle Tyr Ser 180 185 190 Asp Pro Cys Leu Cys Ile Phe Thr Ala Ser Thr AlaTyr Thr Ile Val 195 200 205 Ser Ser Leu Ile Ser Phe Tyr Ile Pro Leu LeuIle Met Leu Val Phe 210 215 220 Tyr Gly Ile Ile Phe Lys Ala Ala Arg AspGln Ala Arg Lys Ile Asn 225 230 235 240 Ala Leu Glu Gly Arg Leu Glu GlnGlu Asn Asn Arg Gly Lys Lys Ile 245 250 255 Ser Leu Ala Lys Glu Lys LysAla Ala Lys Thr Leu Gly Ile Ile Met 260 265 270 Gly Val Phe Ile Leu CysTrp Leu Pro Phe Phe Val Val Asn Ile Val 275 280 285 Asn Pro Phe Cys AspArg Cys Val Gln Pro Ala Val Phe Ile Ala Leu 290 295 300 Thr Trp Leu GlyTrp Ile Asn Ser Cys Phe Asn Pro Ile Ile Tyr Ala 305 310 315 320 Phe AsnLys Glu Phe Arg Lys Val Phe Val Lys Met Ile Cys Cys His 325 330 335 LysCys Arg Gly Val Thr Val Gly Pro Asn His Ala Asp Leu Asn Tyr 340 345 350Asp Pro Val Ala Met Arg Leu Lys Lys Arg Gly Glu Asn Ala Asn Gly 355 360365 Thr Val Asn Gly Asp Ala Asn Gly Lys Ala Asn Gly Asn Ile Glu Ala 370375 380 Gly Glu Gly Thr Ser Ser Ser 385 390 <210> SEQ ID NO 26 <211>LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <214>FEATURE: <215> OTHER INFORMATION: Description of Artificial Sequence:Synthesized peptide <400> SEQUENCE: 26 Met Thr Ser Thr Cys Thr Asn SerThr Arg Glu Ser Asn Ser Ser His 1 5 10 15 Thr Cys Met Pro Leu Ser LysMet Pro Ile Ser Leu Ala His Gly Ile 20 25 30 Ile Arg Ser Thr 35 <210>SEQ ID NO 27 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthesized peptide <400> SEQUENCE: 27 Gln Arg LysPro Gln Leu Leu Gln Val Thr Asn Arg Phe 1 5 10 <210> SEQ ID NO 28 <211>LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthesized peptide <400> SEQUENCE: 28 Trp Pro Leu Asn Ser 1 5 <210> SEQID NO 29 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthesized peptide <400> SEQUENCE: 29 Asp Arg TyrLeu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met 1 5 10 15 Thr GlnArg Arg 20 <210> SEQ ID NO 30 <211> LENGTH: 23 <212> TYPE: PRT <213>ORGANISM: Artifici al Sequence FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthesized peptide <400> SEQUENCE:30 Gly Gln Ala Ala Phe Asp Glu Arg Asn Ala Leu Cys Ser Met Ile Trp 1 510 15 Gly Ala Ser Pro Ser Tyr Thr 20 <210> SEQ ID NO 31 <211> LENGTH:182 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: Synthesizedpeptide <400> SEQUENCE: 31 Cys Ala Ala Arg Arg Gln His Ala Leu Leu TyrAsn Val Lys Arg His 1 5 10 15 Ser Leu Glu Val Arg Val Lys Asp Cys ValGlu Asn Glu Asp Glu Glu 20 25 30 Gly Ala Glu Lys Lys Glu Glu Phe Gln AspGlu Ser Glu Phe Arg Arg 35 40 45 Gln His Glu Gly Glu Val Lys Ala Lys GluGly Arg Met Glu Ala Lys 50 55 60 Asp Gly Ser Leu Lys Ala Lys Glu Gly SerThr Gly Thr Ser Glu Ser 65 70 75 80 Ser Val Glu Ala Gly Ser Glu Glu ValArg Glu Ser Ser Thr Val Ala 85 90 95 Ser Asp Gly Ser Met Glu Gly Lys GluGly Ser Thr Lys Val Glu Glu 100 105 110 Asn Ser Met Lys Ala Asp Lys GlyArg Thr Glu Val Asn Gln Cys Ser 115 120 125 Ile Asp Leu Gly Glu Asp AspMet Glu Phe Gly Glu Asp Asp Ile Asn 130 135 140 Phe Ser Glu Asp Asp ValGlu Ala Val Asn Ile Pro Glu Ser Leu Pro 145 150 155 160 Pro Ser Arg ArgAsn Ser Asn Ser Asn Pro Pro Leu Pro Arg Cys Tyr 165 170 175 Gln Cys LysAla Ala Lys 180 <210> SEQ ID NO 32 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthesized peptide<400> SEQUENCE: 32 Ala Val Leu Ala Val Trp Val Asp Val Glu Thr Gln ValPro Gln 1 5 10 15 <210> SEQ ID NO 33 <211> LENGTH: 55 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthesized peptide<400> SEQUENCE: 33 Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu Ile GlnAsp Met Leu 1 5 10 15 Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys GluAsp Ser His Pro 20 25 30 Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly LysIle Val Pro Ser 35 40 45 Tyr Asp Ser Ala Thr Phe Pro 50 55 <210> SEQ IDNO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: HGPRBMY8 sense primer <400> SEQUENCE: 34 gcagagcactcctccactct 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: HGPRBMY8 anti-sense primer <400>SEQUENCE: 35 agcaggcaat catgacaatc 20 <210> SEQ ID NO 36 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: GPCR84sense primer <400> SEQUENCE: 36 gttagcctca cccacctgtt 20 <210> SEQ ID NO37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: GPCR84 anti-sense primer <400> SEQUENCE: 37 cacaatccaggtgccataga 20 <210> SEQ ID NO 38 <211> LENGTH: 42 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: HGPRBMY8 5′ primer <400> SEQUENCE:38 gtccccaagc ttgcaccatg acgtccacct gcaccaacag ca 42 <210> SEQ ID NO 39<211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: HGPRBMY8 3′ Flag-tag primer <400> SEQUENCE: 39 cgggatcctacttgtcgtcg tcgtccttgt agtccatagg aaaagtagca gaatcgtagg 60 aa 62 <210>SEQ ID NO 40 <211> LENGTH: 407 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 40 Met Ser Leu Asn Ser Ser Leu Ser Cys Arg LysGlu Leu Ser Asn Leu 1 5 10 15 Thr Glu Glu Glu Gly Gly Glu Gly Gly ValIle Ile Thr Gln Phe Ile 20 25 30 Ala Ile Ile Val Ile Thr Ile Phe Val CysLeu Gly Asn Leu Val Ile 35 40 45 Val Val Thr Leu Tyr Lys Lys Ser Tyr LeuLeu Thr Leu Ser Asn Lys 50 55 60 Phe Val Phe Ser Leu Thr Leu Ser Asn PheLeu Leu Ser Val Leu Val 65 70 75 80 Leu Pro Phe Val Val Thr Ser Ser IleArg Arg Glu Trp Ile Phe Gly 85 90 95 Val Val Trp Cys Asn Phe Ser Ala LeuLeu Tyr Leu Leu Ile Ser Ser 100 105 110 Ala Ser Met Leu Thr Leu Gly ValIle Ala Ile Asp Arg Tyr Tyr Ala 115 120 125 Val Leu Tyr Pro Met Val TyrPro Met Lys Ile Thr Gly Asn Arg Ala 130 135 140 Val Met Ala Leu Val TyrIle Trp Leu His Ser Leu Ile Gly Cys Leu 145 150 155 160 Pro Pro Leu PheGly Trp Ser Ser Val Glu Phe Asp Glu Phe Lys Trp 165 170 175 Met Cys ValAla Ala Trp His Arg Glu Pro Gly Tyr Thr Ala Phe Trp 180 185 190 Gln IleTrp Cys Ala Leu Phe Pro Phe Leu Val Met Leu Val Cys Tyr 195 200 205 GlyPhe Ile Phe Arg Val Ala Arg Val Lys Ala Arg Lys Val His Cys 210 215 220Gly Thr Val Val Ile Val Glu Glu Asp Ala Gln Arg Thr Gly Arg Lys 225 230235 240 Asn Ser Ser Thr Ser Thr Ser Ser Ser Gly Ser Arg Arg Asn Ala Phe245 250 255 Gln Gly Val Val Tyr Ser Ala Asn Gln Cys Lys Ala Leu Ile ThrIle 260 265 270 Leu Val Val Leu Gly Ala Phe Met Val Thr Trp Gly Pro TyrMet Val 275 280 285 Val Ile Ala Ser Glu Ala Leu Trp Gly Lys Ser Ser ValSer Pro Ser 290 295 300 Leu Glu Thr Trp Ala Thr Trp Leu Ser Phe Ala SerAla Val Cys His 305 310 315 320 Pro Leu Ile Tyr Gly Leu Trp Asn Lys ThrVal Arg Lys Glu Leu Leu 325 330 335 Gly Met Cys Phe Gly Asp Arg Tyr TyrArg Glu Pro Phe Val Gln Arg 340 345 350 Gln Arg Thr Ser Arg Leu Phe SerIle Ser Asn Arg Ile Thr Asp Leu 355 360 365 Gly Leu Ser Pro His Leu ThrAla Leu Met Ala Gly Gly Gln Pro Leu 370 375 380 Gly His Ser Ser Ser ThrGly Asp Thr Gly Phe Ser Cys Ser Gln Asp 385 390 395 400 Ser Gly Asn LeuArg Ala Leu 405 <210> SEQ ID NO 41 <211> LENGTH: 448 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 41 Met Thr Ser Thr Cys ThrAsn Ser Thr Arg Glu Ser Asn Ser Ser His 1 5 10 15 Thr Cys Met Pro LeuSer Lys Met Pro Ile Ser Leu Ala His Gly Ile 20 25 30 Ile Arg Ser Thr ValLeu Val Ile Phe Leu Ala Ala Ser Phe Val Gly 35 40 45 Asn Ile Val Leu AlaLeu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln 50 55 60 Val Thr Asn Arg PheIle Phe Asn Leu Leu Val Thr Asp Leu Leu Gln 65 70 75 80 Ile Ser Leu ValAla Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe 85 90 95 Trp Pro Leu AsnSer His Phe Cys Thr Ala Leu Val Ser Leu Thr His 100 105 110 Leu Phe AlaPhe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp 115 120 125 Arg TyrLeu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr 130 135 140 GlnArg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile 145 150 155160 Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp 165170 175 Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr180 185 190 Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile ValMet 195 200 205 Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg GlnHis Ala 210 215 220 Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val ArgVal Lys Asp 225 230 235 240 Cys Val Glu Asn Glu Asp Glu Glu Gly Ala GluLys Lys Glu Glu Phe 245 250 255 Gln Asp Glu Ser Glu Phe Arg Arg Gln HisGlu Gly Glu Val Lys Ala 260 265 270 Lys Glu Gly Arg Met Glu Ala Lys AspGly Ser Leu Lys Ala Lys Glu 275 280 285 Gly Ser Thr Gly Thr Ser Glu SerSer Val Glu Ala Arg Gly Ser Glu 290 295 300 Glu Val Arg Glu Ser Ser ThrVal Ala Ser Asp Gly Ser Met Glu Gly 305 310 315 320 Lys Glu Gly Ser ThrLys Val Glu Glu Asn Ser Met Lys Ala Asp Lys 325 330 335 Gly Arg Thr GluVal Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp 340 345 350 Met Glu PheGly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu 355 360 365 Ala ValAsn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn 370 375 380 SerAsn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Lys Lys Val 385 390 395400 Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys 405410 415 Phe Leu Ala Val Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly420 425 430 Gly Thr Glu Gly Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr PhePro 435 440 445 <210> SEQ ID NO 42 <211> LENGTH: 448 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 42 Met Thr Ser Thr Cys ThrAsn Ser Thr Arg Glu Ser Asn Ser Ser His 1 5 10 15 Thr Cys Met Pro LeuSer Lys Met Pro Ile Ser Leu Ala His Gly Ile 20 25 30 Ile Arg Ser Thr ValLeu Val Ile Phe Leu Ala Ala Ser Phe Val Gly 35 40 45 Asn Ile Val Leu AlaLeu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln 50 55 60 Val Thr Asn Arg PheIle Phe Asn Leu Leu Val Thr Asp Leu Leu Gln 65 70 75 80 Ile Ser Leu ValAla Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe 85 90 95 Trp Pro Leu AsnSer His Phe Cys Thr Ala Leu Val Ser Leu Thr His 100 105 110 Leu Phe AlaPhe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp 115 120 125 Arg TyrLeu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr 130 135 140 GlnArg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile 145 150 155160 Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp 165170 175 Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr180 185 190 Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile ValMet 195 200 205 Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg GlnHis Ala 210 215 220 Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val ArgVal Lys Asp 225 230 235 240 Cys Val Glu Asn Glu Asp Glu Glu Gly Ala GluLys Lys Glu Glu Phe 245 250 255 Gln Asp Glu Ser Glu Phe Arg Arg Gln HisGlu Gly Glu Val Lys Ala 260 265 270 Lys Glu Gly Arg Met Glu Ala Lys AspGly Ser Leu Lys Ala Lys Glu 275 280 285 Gly Ser Thr Gly Thr Ser Glu SerSer Val Glu Ala Arg Gly Ser Glu 290 295 300 Glu Val Arg Glu Ser Ser ThrVal Ala Ser Asp Gly Ser Met Glu Gly 305 310 315 320 Lys Glu Gly Ser ThrLys Val Glu Glu Asn Ser Met Lys Ala Asp Lys 325 330 335 Gly Arg Thr GluVal Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp 340 345 350 Met Glu PheGly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu 355 360 365 Ala ValAsn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn 370 375 380 SerAsn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Lys Lys Val 385 390 395400 Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys 405410 415 Phe Leu Ala Val Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly420 425 430 Gly Thr Glu Gly Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr PhePro 435 440 445 <210> SEQ ID NO 43 <211> LENGTH: 448 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 43 Met Thr Ser Thr Cys ThrAsn Ser Thr Arg Glu Ser Asn Ser Ser His 1 5 10 15 Thr Cys Met Pro LeuSer Lys Met Pro Ile Ser Leu Ala His Gly Ile 20 25 30 Ile Arg Ser Thr ValLeu Val Ile Phe Leu Ala Ala Ser Phe Val Gly 35 40 45 Asn Ile Val Leu AlaLeu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln 50 55 60 Val Thr Asn Arg PheIle Phe Asn Leu Leu Val Thr Asp Leu Leu Gln 65 70 75 80 Ile Ser Leu ValAla Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe 85 90 95 Trp Pro Leu AsnSer His Phe Cys Thr Ala Leu Val Ser Leu Thr His 100 105 110 Leu Phe AlaPhe Ala Ser Val Asn Thr Ile Val Leu Val Ser Val Asp 115 120 125 Arg TyrLeu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr 130 135 140 GlnArg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile 145 150 155160 Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp 165170 175 Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr180 185 190 Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile ValMet 195 200 205 Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg GlnHis Ala 210 215 220 Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val ArgVal Lys Asp 225 230 235 240 Cys Val Glu Asn Glu Asp Glu Glu Gly Ala GluLys Lys Glu Glu Phe 245 250 255 Gln Asp Glu Ser Glu Phe Arg Arg Gln HisGlu Gly Glu Val Lys Ala 260 265 270 Lys Glu Gly Arg Met Glu Ala Lys AspGly Ser Leu Lys Ala Lys Glu 275 280 285 Gly Ser Thr Gly Thr Ser Glu SerSer Val Glu Ala Arg Gly Ser Glu 290 295 300 Glu Val Arg Glu Ser Ser ThrVal Ala Ser Asp Gly Ser Met Glu Gly 305 310 315 320 Lys Glu Gly Ser ThrLys Val Glu Glu Asn Ser Met Lys Ala Asp Lys 325 330 335 Gly Arg Thr GluVal Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Gly 340 345 350 Met Glu PheGly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu 355 360 365 Ala ValAsn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn 370 375 380 SerAsn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Ala Lys Val 385 390 395400 Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys 405410 415 Phe Leu Ala Val Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly420 425 430 Gly Thr Glu Gly Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr PhePro 435 440 445 <210> SEQ ID NO 44 <211> LENGTH: 1659 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 44 gcctgcaacc tgtcycacgccctctggctg ttgccatgac gtccacctgc accaacagca 60 cgcgcgagag taacagcagccacacgtgca tgcccctctc caaaatgccc atcagcctgg 120 cccacggcat catccgctcaaccgtgctgg ttatcttcct cgccgcctct ttcgtcggca 180 acatagtgct ggcgctagtgttgcagcgca agccgcagct gctgcaggtg accaaccgtt 240 ttatctttaa cctcctcgtcaccgacctgc tgcagatttc gctcgtggcc ccctgggtgg 300 tggccacctc tgtgcctctcttctggcccc tcaacagcca cttctgcacg gccctggtta 360 gcctcaccca cctgttcgccttcgccagcg tcaacaccat tgtcttggtg tcagtggatc 420 gctacttgtc catcatccaccctctctcct acccgtccaa gatgacccag cgccgcggtt 480 acctgctcct ctatggcacctggattgtgg ccatcctgca gagcactcct ccactctacg 540 gctggggcca ggctgcctttgatgagcgca atgctctctg ctccatgatc tggggggcca 600 gccccagcta cactattctcagcgtggtgt ccttcatcgt cattccactg attgtcatga 660 ttgcctgcta ctccgtggtgttctgtgcag cccggaggca gcatgctctg ctgtacaatg 720 tcaagagaca cagcttggaagtgcgagtca aggactgtgt ggagaatgag gatgaagagg 780 gagcagagaa gaaggaggagttccaggatg agagtgagtt tcgccgccag catgaaggtg 840 aggtcaaggc caaggagggcagaatggaag ccaaggacgg cagcctgaag gccaaggaag 900 gaagcacggg gaccagtgagagtagtgtag aggccagggg cagcgaggag gtcagagaga 960 gcagcacggt ggccagcgacggcagcatgg agggtaagga aggcagcacc aaagttgagg 1020 agaacagcat gaaggcagacaagggtcgca cagaggtcaa ccagtgcagc attgacttgg 1080 gtgaagatgg catggagtttggtgaagacg acatcaattt cagtgaggat gacgtcgagg 1140 cagtgaacat cccggagagcctcccaccca gtcgtcgtaa cagcaacagc aaccctcctc 1200 tgcccaggtg ctaccagtgcaaagctgcta aagtgatctt catcatcatt ttctcctatg 1260 tgctatccct ggggccctactgctttttag cagtcctggc cgtgtgggtg gatgtcgaaa 1320 cccaggtacc ccagtgggtgatcaccataa tcatctggct tttcttcctg cagtgctgca 1380 tccaccccta tgtctatggctacatgcaca agaccattaa gaaggaaatc caggacatgc 1440 tgaagaagtt cttctgcaaggaaaagcccc cgaaagaaga tagccaccca gacctgcccg 1500 gaacagaggg tgggactgaaggcaagattg tcccttccta cgattctgct acttttcctt 1560 gaagttagtt ctaaggcaaaccttgaaaat cagtccttca gccacagcta tttagagctt 1620 taaaactacc aggttcaatcactggttatg ctttctgtg 1659 <210> SEQ ID NO 45 <211> LENGTH: 1527 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 45 atgacgtccacctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc 60 ctctccaaaatgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120 ttcctcgccgcctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180 cagctgctgcaggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240 atttcgctcgtggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300 agccacttctgcacggccct ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360 accattgtcgtggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420 tccaagatgacccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480 ctgcagagcactcctccact ctacggctgg ggccaggctg cctttgatga gcgcaatgct 540 ctctgctccatgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600 atcgtcattccactgattgt catgattgcc tgctactccg tggtgttctg tgcagcccgg 660 aggcagcatgctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720 tgtgtggagaatgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780 gagtttcgccgccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840 gacggcagcctgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900 aggggcagcgaggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960 aaggaaggcagcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020 gtcaaccagtgcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080 aatttcagtgaggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140 cgtaacagcaacagcaaccc tcctctgccc aggtgctacc agtgcaaagc tgctaaagtg 1200 atcttcatcatcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260 ctggccgtgtgggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320 tggcttttcttcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380 attaagaaggaaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440 gaagatagccacccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500 tcctacgattctgctacttt tccttga 1527 <210> SEQ ID NO 46 <211> LENGTH: 1527 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 46 atgacgtccacctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc 60 ctctccaaaatgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120 ttcctcgccgcctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180 cagctgctgcaggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240 atttcgctcgtggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300 agccacttctgcacggccct ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360 accattgtcgtggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420 tccaagatgacccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480 ctgcagagcactcctccact ctacggctgg ggccaggctg cctttgatga gcgcaatgct 540 ctctgctccatgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600 atcgtcattccactgattgt catgattgcc tgctactccg tggtgttctg tgcagcccgg 660 aggcagcatgctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720 tgtgtggagaatgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780 gagtttcgccgccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840 gacggcagcctgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900 aggggcagcgaggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960 aaggaaggcagcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020 gtcaaccagtgcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080 aatttcagtgaggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140 cgtaacagcaacagcaaccc tcctctgccc aggtgctacc agtgcaaagc taagaaagtg 1200 atcttcatcatcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260 ctggccgtgtgggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320 tggcttttcttcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380 attaagaaggaaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440 gaagatagccacccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500 tcctacgattctgctacttt tccttga 1527 <210> SEQ ID NO 47 <211> LENGTH: 1580 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 47 gcaacctgtctcacgccctc tggctgttgc catgacgtcc acctgcacca acagcacgcg 60 cgagagtaacagcagccaca cgtgcatgcc cctctccaaa atgcccatca gcctggccca 120 cggcatcatccgctcaaccg tgctggttat cttcctcgcc gcctctttcg tcggcaacat 180 agtgctggcgctagtgttgc agcgcaagcc gcagctgctg caggtgacca accgttttat 240 ctttaacctcctcgtcaccg acctgctgca gatttcgctc gtggccccct gggtggtggc 300 cacctctgtgcctctcttct ggcccctcaa cagccacttc tgcacggccc tggttagcct 360 cacccacctgttcgccttcg ccagcgtcaa caccattgtc ttggtgtcag tggatcgcta 420 cttgtccatcatccaccctc tctcctaccc gtccaagatg acccagcgcc gcggttacct 480 gctcctctatggcacctgga ttgtggccat cctgcagagc actcctccac tctacggctg 540 gggccaggctgcctttgatg agcgcaatgc tctctgctcc atgatctggg gggccagccc 600 cagctacactattctcagcg tggtgtcctt catcgtcatt ccactgattg tcatgattgc 660 ctgctactccgtggtgttct gtgcagcccg gaggcagcat gctctgctgt acaatgtcaa 720 gagacacagcttggaagtgc gagtcaagga ctgtgtggag aatgaggatg aagagggagc 780 agagaagaaggaggagttcc aggatgagag tgagtttcgc cgccagcatg aaggtgaggt 840 caaggccaaggagggcagaa tggaagccaa ggacggcagc ctgaaggcca aggaaggaag 900 cacggggaccagtgagagta gtgtagaggc caggggcagc gaggaggtca gagagagcag 960 cacggtggccagcgacggca gcatggaggg taaggaaggc agcaccaaag ttgaggagaa 1020 cagcatgaaggcagacaagg gtcgcacaga ggtcaaccag tgcagcattg acttgggtga 1080 agatgacatggagtttggtg aagacgacat caatttcagt gaggatgacg tcgaggcagt 1140 gaacatcccggagagcctcc cacccagtcg tcgtaacagc aacagcaacc ctcctctgcc 1200 caggtgctaccagtgcaaag ctgctaaagt gatcttcatc atcattttct cctatgtgct 1260 atccctggggccctactgct ttttagcagt cctggccgtg tgggtggatg tcgaaaccca 1320 ggtaccccagtgggtgatca ccataatcat ctggcttttc ttcctgcagt gctgcatcca 1380 cccctatgtctatggctaca tgcacaagac cattaagaag gaaatccagg acatgctgaa 1440 gaagttcttctgcaaggaaa agcccccgaa agaagatagc cacccagacc tgcccggaac 1500 agagggtgggactgaaggca agattgtccc ttcctacgat tctgctactt ttccttgaag 1560 ttagttctaaggcaaacctt 1580 <210> SEQ ID NO 48 <211> LENGTH: 1527 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:modified_base <222> LOCATION: (1192)..(1194) <223> OTHER INFORMATION:n=a or t or g or c <400> SEQUENCE: 48 atgacgtcca cctgcaccaa cagcacgcgcgagagtaaca gcagccacac gtgcatgccc 60 ctctccaaaa tgcccatcag cctggcccacggcatcatcc gctcaaccgt gctggttatc 120 ttcctcgccg cctctttcgt cggcaacatagtgctggcgc tagtgttgca gcgcaagccg 180 cagctgctgc aggtgaccaa ccgttttatctttaacctcc tcgtcaccga cctgctgcag 240 atttcgctcg tggccccctg ggtggtggccacctctgtgc ctctcttctg gcccctcaac 300 agccacttct gcacggccct ggttagcctcacccacctgt tcgccttcgc cagcgtcaac 360 accattgtcn tggtgtcagt ggatcgctacttgtccatca tccaccctct ctcctacccg 420 tccaagatga cccagcgccg cggttacctgctcctctatg gcacctggat tgtggccatc 480 ctgcagagca ctcctccact ctacggctggggccaggctg cctttgatga gcgcaatgct 540 ctctgctcca tgatctgggg ggccagccccagctacacta ttctcagcgt ggtgtccttc 600 atcgtcattc cactgattgt catgattgcctgctactccg tggtgttctg tgcagcccgg 660 aggcagcatg ctctgctgta caatgtcaagagacacagct tggaagtgcg agtcaaggac 720 tgtgtggaga atgaggatga agagggagcagagaagaagg aggagttcca ggatgagagt 780 gagtttcgcc gccagcatga aggtgaggtcaaggccaagg agggcagaat ggaagccaag 840 gacggcagcc tgaaggccaa ggaaggaagcacggggacca gtgagagtag tgtagaggcc 900 aggggcagcg aggaggtcag agagagcagcacggtggcca gcgacggcag catggagggt 960 aaggaaggca gcaccaaagt tgaggagaacagcatgaagg cagacaaggg tcgcacagag 1020 gtcaaccagt gcagcattga cttgggtgaagatgncatgg agtttggtga agacgacatc 1080 aatttcagtg aggatgacgt cgaggcagtgaacatcccgg agagcctccc acccagtcgt 1140 cgtaacagca acagcaaccc tcctctgcccaggtgctacc agtgcaaagc tnnnaaagtg 1200 atcttcatca tcattttctc ctatgtgctatccctggggc cctactgctt tttagcagtc 1260 ctggccgtgt gggtggatgt cgaaacccaggtaccccagt gggtgatcac cataatcatc 1320 tggcttttct tcctgcagtg ctgcatccacccctatgtct atggctacat gcacaagacc 1380 attaagaagg aaatccagga catgctgaagaagttcttct gcaaggaaaa gcccccgaaa 1440 gaagatagcc acccagacct gcccggaacagagggtggga ctgaaggcaa gattgtccct 1500 tcctacgatt ctgctacttt tccttga 1527<210> SEQ ID NO 49 <211> LENGTH: 508 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION:(124) <223> OTHER INFORMATION: Xaa=Unknown, modified, or any amino acid<221> NAME/KEY: VARIANT <222> LOCATION: (398) <223> OTHER INFORMATION:Xaa=Unknown, modified, or any amino acid <400> SEQUENCE: 49 Met Thr SerThr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His 1 5 10 15 Thr CysMet Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly Ile 20 25 30 Ile ArgSer Thr Val Leu Val Ile Phe Leu Ala Ala Ser Phe Val Gly 35 40 45 Asn IleVal Leu Ala Leu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln 50 55 60 Val ThrAsn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp Leu Leu Gln 65 70 75 80 IleSer Leu Val Ala Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe 85 90 95 TrpPro Leu Asn Ser His Phe Cys Thr Ala Leu Val Ser Leu Thr His 100 105 110Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val Xaa Val Ser Val Asp 115 120125 Arg Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr 130135 140 Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile145 150 155 160 Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala AlaPhe Asp 165 170 175 Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala SerPro Ser Tyr 180 185 190 Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile ProLeu Ile Val Met 195 200 205 Ile Ala Cys Tyr Ser Val Val Phe Cys Ala AlaArg Arg Gln His Ala 210 215 220 Leu Leu Tyr Asn Val Lys Arg His Ser LeuGlu Val Arg Val Lys Asp 225 230 235 240 Cys Val Glu Asn Glu Asp Glu GluGly Ala Glu Lys Lys Glu Glu Phe 245 250 255 Gln Asp Glu Ser Glu Phe ArgArg Gln His Glu Gly Glu Val Lys Ala 260 265 270 Lys Glu Gly Arg Met GluAla Lys Asp Gly Ser Leu Lys Ala Lys Glu 275 280 285 Gly Ser Thr Gly ThrSer Glu Ser Ser Val Glu Ala Arg Gly Ser Glu 290 295 300 Glu Val Arg GluSer Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly 305 310 315 320 Lys GluGly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys 325 330 335 GlyArg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Xaa 340 345 350Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu 355 360365 Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn 370375 380 Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Xaa Lys Val385 390 395 400 Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly ProTyr Cys 405 410 415 Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu ThrGln Val Pro 420 425 430 Gln Trp Val Ile Thr Ile Ile Ile Trp Leu Phe PheLeu Gln Cys Cys 435 440 445 Ile His Pro Tyr Val Tyr Gly Tyr Met His LysThr Ile Lys Lys Glu 450 455 460 Ile Gln Asp Met Leu Lys Lys Phe Phe CysLys Glu Lys Pro Pro Lys 465 470 475 480 Glu Asp Ser His Pro Asp Leu ProGly Thr Glu Gly Gly Thr Glu Gly 485 490 495 Lys Ile Val Pro Ser Tyr AspSer Ala Thr Phe Pro 500 505 <210> SEQ ID NO 50 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 50caccattgtc ttggtgtcag t 21 <210> SEQ ID NO 51 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 51caccattgtc gtggtgtcag t 21 <210> SEQ ID NO 52 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 52ggtgaagatg acatggagtt t 21 <210> SEQ ID NO 53 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 53ggtgaagatg gcatggagtt t 21 <210> SEQ ID NO 54 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 54gtgcaaagct gctaaagtga t 21 <210> SEQ ID NO 55 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 55gtgcaaagct actaaagtga t 21 <210> SEQ ID NO 56 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 56tgcaaagctg ctaaagtgat c 21 <210> SEQ ID NO 57 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 57tgcaaagctg ataaagtgat c 21 <210> SEQ ID NO 58 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 58gcaaagctgc taaagtgatc t 21 <210> SEQ ID NO 59 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SNP <400> SEQUENCE: 59gcaaagctgc gaaagtgatc t 21 <210> SEQ ID NO 60 <211> LENGTH: 17 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: GAPDH F3 Forward primer<400> SEQUENCE: 60 agccgagcca catcgct 17 <210> SEQ ID NO 61 <211>LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:GAPDH R1 Reverse primer <400> SEQUENCE: 61 gtgaccaggc gcccaatac 19 <210>SEQ ID NO 62 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: GAPDH-PVIC Taqman(R) Probe <400> SEQUENCE: 62caaatccgtt gactccgacc ttcacctt 28 <210> SEQ ID NO 63 <211> LENGTH: 99<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide 1<221> NAME/KEY: modified_base <222> LOCATION: (25)..(84) <223> OTHERINFORMATION: n=a+g+c+t and b=c+g+t <400> SEQUENCE: 63 cgaagcgtaagggcccagcc ggccnnbnnb nnbnnbnnbn nbnnbnnbnn bnnbnnbnnb 60 nnbnnbnnbnnbnnbnnbnn bnnbccgggt ccgggcggc 99 <210> SEQ ID NO 64 <211> LENGTH: 95<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide2N=A+G+C+T and V=C+A+G <221> NAME/KEY: modified_base <222> LOCATION:(21)..(80) <223> OTHER INFORMATION: n=a or g or c or t; v=c or a or g<400> SEQUENCE: 64 aaaaggaaaa aagcggccgc vnnvnnvnnv nnvnnvnnvnnvnnvnnvnn vnnvnnvnnv 60 nnvnnvnnvn nvnnvnnvnn gccgcccgga cccgg 95 <210>SEQ ID NO 65 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic polypeptide <400> SEQUENCE: 65 Pro GlyPro Gly Gly 1 5 <210> SEQ ID NO 66 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 66 Gly Asp Phe Trp Tyr Glu Ala Cys Glu Ser Ser Cys AlaPhe Trp 1 5 10 15 <210> SEQ ID NO 67 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 67 Leu Glu Trp Gly Ser Asp Val Phe Tyr Asp Val Tyr AspCys Cys 1 5 10 15 <210> SEQ ID NO 68 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 68 Cys Leu Arg Ser Gly Thr Gly Cys Ala Phe Gln Leu TyrArg Phe 1 5 10 15 <210> SEQ ID NO 69 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 69 Asn Asn Phe Pro Cys Leu Arg Ser Gly Arg Asn Cys AspAla Gly 1 5 10 15 <210> SEQ ID NO 70 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 70 Arg Ile Val Pro Asn Gly Tyr Phe Asn Val His Gly ArgSer Leu 1 5 10 15 <210> SEQ ID NO 71 <211> LENGTH: 14 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 71 Arg Ile Asp Ser Cys Ala Lys Tyr Phe Leu Arg Ser CysAsp 1 5 10 <210> SEQ ID NO 72 <211> LENGTH: 39 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic 5′ primer <400> SEQUENCE:72 gcagcagcgg ccgcaccgtg ctggttatct tcctcgccg 39 <210> SEQ ID NO 73<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic 3′ primer <400> SEQUENCE: 73 gcagcagtcg acaggaaaagtagcagaatc gtagg 35 <210> SEQ ID NO 74 <211> LENGTH: 38 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic 5′ primer<400> SEQUENCE: 74 gcagcagcgg ccgcatgacg tccacctgca ccaacagc 38 <210>SEQ ID NO 75 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic 3′ primer <400> SEQUENCE: 75 gcagcagtcgacatagacat aggggtggat gcagcac 37 <210> SEQ ID NO 76 <211> LENGTH: 13<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Syntheticpolypeptide <400> SEQUENCE: 76 Ser Thr Cys Thr Asn Ser Thr Arg Glu SerAsn Ser Ser 1 5 10 <210> SEQ ID NO 77 <211> LENGTH: 13 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 77 Gln Leu Leu Gln Val Thr Asn Arg Phe Ile Phe Asn Leu 15 10 <210> SEQ ID NO 78 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 78 Tyr ProSer Lys Met Thr Gln Arg Arg Gly Tyr Leu Leu 1 5 10 <210> SEQ ID NO 79<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic polypeptide <400> SEQUENCE: 79 Glu Ala Lys Asp GlySer Leu Lys Ala Lys Glu Gly Ser 1 5 10 <210> SEQ ID NO 80 <211> LENGTH:13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: Syntheticpolypeptide <400> SEQUENCE: 80 Glu Gly Lys Glu Gly Ser Thr Lys Val GluGlu Asn Ser 1 5 10 <210> SEQ ID NO 81 <211> LENGTH: 13 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 81 Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys Gly Arg 15 10 <210> SEQ ID NO 82 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 82 Glu SerLeu Pro Pro Ser Arg Arg Asn Ser Asn Ser Asn 1 5 10 <210> SEQ ID NO 83<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic polypeptide <400> SEQUENCE: 83 Gly Tyr Met His LysThr Ile Lys Lys Glu Ile Gln Asp 1 5 10 <210> SEQ ID NO 84 <211> LENGTH:14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: Syntheticpolypeptide <400> SEQUENCE: 84 Ser Thr Cys Thr Asn Ser Thr Arg Glu SerAsn Ser Ser His 1 5 10 <210> SEQ ID NO 85 <211> LENGTH: 14 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 85 Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly SerGlu 1 5 10 <210> SEQ ID NO 86 <211> LENGTH: 14 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic polypeptide <400>SEQUENCE: 86 Gly Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys 1 510 <210> SEQ ID NO 87 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 87 Asp AspIle Asn Phe Ser Glu Asp Asp Val Glu Ala Val Asn 1 5 10 <210> SEQ ID NO88 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic polypeptide <400> SEQUENCE: 88 Pro Pro Lys Glu AspSer His Pro Asp Leu Pro Gly Thr Glu 1 5 10 <210> SEQ ID NO 89 <211>LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 89 Leu Leu Tyr Asn Val Lys Arg HisSer Leu Glu Val Arg Val 1 5 10 <210> SEQ ID NO 90 <211> LENGTH: 14 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic polypeptide<400> SEQUENCE: 90 Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn Ser Asn ProPro 1 5 10 <210> SEQ ID NO 91 <211> LENGTH: 14 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic polypeptide <400>SEQUENCE: 91 Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser 1 510 <210> SEQ ID NO 92 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 92 Ser ThrArg Glu Ser Asn Ser Ser His Thr Cys Met Pro Leu 1 5 10 <210> SEQ ID NO93 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic polypeptide <400> SEQUENCE: 93 Gly Glu Asp Asp IleAsn Phe Ser Glu Asp Asp Val Glu Ala 1 5 10 <210> SEQ ID NO 94 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 94 Ile Ser Leu Ala His Gly Ile IleArg Ser Thr Val Leu Val Ile Phe 1 5 10 15 <210> SEQ ID NO 95 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 95 Cys Ser Met Ile Trp Gly Ala SerPro Ser Tyr Thr Ile Leu Ser Val 1 5 10 15 <210> SEQ ID NO 96 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 96 Met Glu Ala Lys Asp Gly Ser LeuLys Ala Lys Glu Gly Ser Thr Gly 1 5 10 15 <210> SEQ ID NO 97 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 97 Leu Lys Ala Lys Glu Gly Ser ThrGly Thr Ser Glu Ser Ser Val Glu 1 5 10 15 <210> SEQ ID NO 98 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 98 Lys Glu Gly Ser Thr Gly Thr SerGlu Ser Ser Val Glu Ala Arg Gly 1 5 10 15 <210> SEQ ID NO 99 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 99 Thr Val Ala Ser Asp Gly Ser MetGlu Gly Lys Glu Gly Ser Thr Lys 1 5 10 15 <210> SEQ ID NO 100 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 100 His Pro Asp Leu Pro Gly ThrGlu Gly Gly Thr Glu Gly Lys Ile Val 1 5 10 15 <210> SEQ ID NO 101 <211>LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 101 Leu Pro Gly Thr Glu Gly GlyThr Glu Gly Lys Ile Val Pro Ser Tyr 1 5 10 15 <210> SEQ ID NO 102 <211>LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic polypeptide <400> SEQUENCE: 102 Ser Val Val Ser Phe Ile ValIle Pro Leu Ile Val Met Ile Ala Cys 1 5 10 15 Tyr Ser Val Val Phe 20

What is claimed is:
 1. An isolated polynucleotide selected from thegroup consisting of: (a) an isolated polynucleotide encoding a humanG-protein coupled receptor, or functional fragment thereof, comprisingthe amino acid sequence as set forth in SEQ ID NO:2; (b) An isolatedcomposition comprising the polynucleotide according to (a). (c) Anisolated polynucleotide comprising SEQ ID NO:1; (d) An isolatedpolynucleotide having the nucleic acid sequence of ATCC Accession No.PTA-2966; (e) An isolated polynucleotide having the nucleic acidsequence according to nucleotides 4 to 1524 of SEQ ID NO:1, wherein saidnucleotides encode a polypeptide of SEQ ID NO:2 minus the start codon;(f) An isolated polynucleotide having the nucleic acid sequenceaccording to nucleotides 1 to 1524 of SEQ ID NO:1, wherein saidnucleotides encode a polypeptide of SEQ ID NO:2 including the startcodon; (g) A polynucleotide which is fully complementary to thepolynucleotide according to (a) thru (f); and (h) A hybridization probecomprising the polynucleotide according to (a) thru (g).
 2. Anexpression vector containing the polynucleotide according to claim
 1. 3.A host cell containing the expression vector according to claim
 2. 4. Asubstantially purified G-protein coupled receptor polypeptide selectedfrom the group consisting of: (a) A substantially purified G-proteincoupled receptor polypeptide comprising an amino acid sequence as setforth in SEQ ID NO:2. (b) The polypeptide according to (a), wherein theamino acid sequence differs from SEQ ID NO:2 only by conservativesubstitutions; (c) An isolated and substantially purified G-proteincoupled receptor polypeptide encoded by the nucleic acid sequence ofATCC Accession No. PTA-2966; (d) An isolated polypeptide having theamino acid sequence according to amino acids 2 to 508 of SEQ ID NO:2,wherein said amino acid encode a polypeptide of SEQ ID NO:2 minus thestart methionine; (e) An isolated polypeptide having the amino acidsequence according to amino acids 1 to 508 of SEQ ID NO:2, wherein saidamino acid encode a polypeptide of SEQ ID NO:2 including the startmethionine; (f) A substantially purified fragment of the G-proteincoupled receptor polypeptide according to any one of (a) to (e).
 5. Asubstantially purified fusion protein comprising an amino acid sequenceas set forth in SEQ ID NO:2 and an amino acid sequence of an Fc portionof a human immunoglobulin protein.
 6. A pharmaceutical compositioncomprising the polypeptide, or a functional fragment thereof, accordingto claim 1, and a pharmaceutically acceptable diluent or excipient.
 7. Apurified antibody which binds specifically to the polypeptide accordingto claim 4, or an antigenic epitope thereof.
 8. A method of screening alibrary of molecules or compounds with a polynucleotide to identify atleast one molecule or compound therein which specifically binds to thepolynucleotide sequence, comprising: (a) combining the polynucleotideaccording to claim 1, with a library of molecules or compounds underconditions to allow specific binding; and (b) detecting specificbinding, thereby identifying a molecule or compound, which specificallybinds to a G-protein coupled receptor-encoding polynucleotide sequence.9. The method according to claim 8, wherein the candidate compounds aresmall molecules, therapeutics, biological agents, or drugs.
 10. A methodof screening for candidate compounds capable of modulating activity of aG-protein coupled receptor-encoding polypeptide, comprising: (a)contacting a test compound with a cell or tissue expressing thepolypeptide according to claim 4; and (b) selecting as candidatemodulating compounds those test compounds that modulate activity of theG-protein coupled receptor polypeptide.
 11. A method of treating aneurological disorder in a mammal comprising administration of theG-protein coupled receptor polypeptide or homologue according to any oneof claims 1, 4, or, 5 in an amount effective to treat the neurologicaldisorder.
 12. A substantially purified G-protein coupled receptorpolypeptide consisting of an amino acid sequence as set forth in SEQ IDNO:2.
 13. The polypeptide according to claim 12, wherein the amino acidsequence differs from SEQ ID NO:2 only by conservative substitutions.14. An isolated and purified polynucleotide encoding a human G-proteincoupled receptor, or functional fragment thereof, consisting of theamino acid sequence as set forth in SEQ ID NO:2.
 15. A method oftreating a disease, disorder, or condition related to the braincomprising administering the G-protein coupled receptor polypeptide orhomologue according to claim 12 or 13 in an amount effective to treatthe brain-related disorder.
 16. The polypeptide of claim 12 or 13,further comprising the polypeptide expressed in the caudate nucleus,substantia nigra, thalamus, amygdala, hippocampus, cerebellum, andcorpus collosum.
 17. A cell comprising NFAT/CRE and the polypeptide ofclaim 12 or
 13. 18. A cell comprising NFAT G alpha 15 and thepolypeptide of claim 12 or
 13. 19. A method of screening for candidatecompounds capable of modulating activity of a G-protein coupledreceptor-encoding polypeptide, comprising: (a) contacting a testcompound with a cell or tissue expressing the polypeptide according toclaim 12 or 13; and (b) selecting as candidate modulating compoundsthose test compounds that modulate activity of the G-protein coupledreceptor polypeptide.
 20. The method according to claim 19, wherein thecandidate compounds are agonists or antagonists of G-protein coupledreceptor activity.
 21. The method according to claim 20, wherein thepolypeptide activity is associated with the brain.
 22. The methodaccording to claim 20, wherein the candidate modulating compounds arepeptides.